Lymphatic zip codes in tumors and pre-malignant lesions

ABSTRACT

Disclosed herein are compositions and methods for and involving selectively targeting tumor lymphatics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/670,318, filed Feb. 1, 2007, which claims benefit of U.S. ProvisionalApplication No. 60/764,175, filed Feb. 1, 2006. U.S. application Ser.No. 11/670,318, filed Feb. 1, 2007, and U.S. Provisional Application No.60/764,175, filed Feb. 1, 2006, are hereby incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grants PO1 CA82713, PO1 CA 104898, P30 CA 30199, RO1 CA 115410 awarded by NationalInstitutes of Health and Grants DAMD 17-02-1-0315, Grant T32 CA77109-05awarded by the National Cancer Institute, and DAMD 17-02-0309 awarded bythe Department of Defense. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Feb. 10, 2011 as a text file named“SBMRI_(—)26_(—)8403 AMD_AFD_Sequence_Listing_Text_File,” created onFeb. 2, 2011, and having a size of 15,594 bytes is hereby incorporatedby reference pursuant to 37 C.F.R. §1.52(e)(5).

BACKGROUND

The endothelial lining of blood vessels is highly diversified. Many,perhaps all, normal tissues put a tissue-specific “signature” on theirvasculature, and tumor vessels differ from normal vessels both inmorphology and molecular composition (Ruoslahti, 2002). Tumors induceangiogenesis to accommodate the growth of the tumor (Hanahan andWeinberg, 2000) and many of the changes in tumor vessels areangiogenesis-related (Brooks et al., 1994; Christian et al., 2003;Ferrara and Alitalo, 1999; Pasqualini et al., 2000). Moreover, tumorblood vessels have tumor type-specific and, in some stages,stage-specific characteristics; in vivo screening of phage librariesyielded distinct sets of homing peptides selectively recognizingangiogenic signatures in two transgenic mouse models of organ specifictumorigenesis. Homing peptides can also distinguish the angiogenic bloodvessels of pre-malignant lesions from those of fully malignant lesionsin the same tumor model (Hoffman et al., 2003; Joyce et al., 2003),indicating that vascular changes mirror the stage of tumor development.

The lymphatic system constitutes a second vascular system, one that hasonly an efferent arm. Tumors frequently induce lymphangiogenesis, aswell as co-opt existing lymphatics (Cao et al., 2004; Cassella andSkobe, 2002; Stacker et al., 2002). Tumors may contain intratumorallymphatics, but, more commonly, an extensive network of lymphaticvessels is present around tumor tissue (Jackson et al., 2001; Laakkonenet al., 2002; Padera et al., 2002). The lymphatics within tumors, whenpresent, are generally non-functional in fluid transport (Padera et al.,2002), possibly reflecting compression by interstitial pressure andblockade by intra-luminal tumor cells. The lymphatic vessels in andaround tumors are an important conduit of metastasis. Indeed, growthfactor-stimulated enhancement of lymphatic vessel expression in tumorsincreases metastasis (Mandriota et al., 2001; Skobe et al., 2001).Conversely, inhibiting lymphangigenesis suppresses lymphatic metastasis,but generally does not affect tumor growth (Saharinen et al., 2004).

As disclosed herein, tumor lymphatics, like tumor blood vessels, expressspecific markers, and that these lymphatic markers are tumortype-specific and distinct from blood vessel markers in the same tumors.Thus, needed in the art are compositions and methods for thatselectively bind tumor lymphatics or lymphatics in pre-malignant lesionsfor use in early detection and tumor targeting.

BRIEF SUMMARY

In accordance with the purpose of this invention, as embodied andbroadly described herein, this invention relates to peptides,compositions, conjugates, nucleic acids and methods for and involvingselectively targeting tumor lymphatics and/or tumors and tumor cells.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 shows a homing peptide recognizes C8161 melanoma lymphatics.Homing of LSD-phage to C8161 xenografts. The LSD phage clone (2×10⁹ pfu)was injected intravenously into mice bearing C8161 xenograft tumors andallowed to circulate for 7 min. Phage titers recovered from tumors andcontrol tissues are shown. Phage accumulation in C8161 tumor tissue wassignificantly higher than in normal tissues. P<0.03 relative to thenormal tissue with the highest phage uptake, the skin; (n=3).

FIGS. 2A and 2B show stage-specific peptides distinguish pre-malignantlesions and tumors in the prostate of TRAMP mice and colocalize withlymphatic vessels. Phage isolated by screening for homing to TRAMPtumors (REA) or to TRAMP pre-malignant lesions (AGR) were individuallytested in TRAMP mice bearing tumors, or pre-malignant lesions, and intumor-free littermates of TRAMP mice with normal prostate. TRAMP micewere intravenously injected with phage or fluorescein-conjugatedpeptides, and the localization of the phage was studied by phagetitration or immunohistochemistry in frozen tissue sections. Thepeptides were detected in tissue sections by examining fluorescence. TheREA-phage (A) accumulate in TRAMP tumors, whereas the AGR phage (B)selectively home to pre-malignant lesions. The difference between tumortissue and pre-malignant tissue was significant for both peptides(P<0.01; n=3 to 6) Original magnification: 400×.

FIG. 3 shows LyP-2 peptide homes to lymphatics in pre-malignant lesionsand tumors of cervix in K14-HPV16/E2 transgenic mice. LyP-2 phage(1.5×10⁹ pfu) was intravenously injected into mice bearing CIN-3 lesionsor tumors of the cervix and phage titers from the indicated tissues weredetermined. Significantly more of the LyP-2 phage accumulated in thetumors and dysplastic lesions than in normal cervix (P<0.005; n=3).

FIG. 4 shows tumor-type specificity of the LSD peptide. In vivo homingof the phage to six types of tumors was tested as in FIG. 1 (n=3 to 6).Robust phage homing was only observed in C8161 tumors. KRIB xenografttumors were slightly positive for phage and peptide homing, but phagehoming to C8161 tumors was significantly higher than to this or any ofthe other tumors (P<0.005).

FIGS. 5A and 5B show homing specificity of the REA and AGR peptides indifferent types of tumors and pre-malignant lesions. In vivo homing ofthe REA-phage (A) to eleven types of tumors was tested (n=3 to 6).Significant phage homing was observed in prostate tumors of TRAMP mice,and in PPC1, M12, DU145 and LNCaP human prostate cancer xenografttumors. Four out of 5 prostate cancers (DU145 was the exception)accumulated significantly more REA phage than the other types of tumors(P<0.03). Cervical tumors in K14-HPV16/E2 mice were slightly positive.FIG. 5B shows in vivo homing of intravenously injected AGR phage inTRAMP mice, K14-HPV16/E2 mice bearing CIN-3 lesions or tumors (n=3), andin MMTV-PyMT mice with dysplastic lesions or breast tumors. The AGRphage homed significantly more to TRAMP pre-malignant lesions than tocomparable lesions in the other tumor models (P<0.03), and the TRAMPlesions are strongly positive for AGR peptide homing. A MMTVPyMTdysplastic breast lesion is weakly positive for AGR peptide binding.

FIG. 6 shows differential tumor-homing specificity of LyP-1 and LyP-2peptides. LyP-1 and LyP-2 phage were intravenously injected into micebearing MDA-MB-435 breast cancer xenografts or K14-HPV16/E2 tumors(n=3). Tissues were collected and processed for histological analysis 2hrs later. LyP-1 homes to the MDA-MB-435 tumors, whereas LyP-2 homes tothe cervical cancers and pre-malignant lesions. Phage homing to thepre-malignant lesions was significantly higher than to the correspondingtumors in both models (P<0.01).

FIGS. 7A, 7B and 7C show transfection with the chemokine receptor CXCR4increases the binding capability for the LSD peptide. FIG. 7A showshomology region with LSD peptide was found in residues 17-22 ofpro-CXCL12 by searching NCBI BLAST against SWISSPROT database. FIG. 7Bshows the LSD phage binds to 293T cells transfected with CXCR4, but notto cells transfected with VEGFR2 or with the empty vector (P<0.01). FIG.7C shows the LSD peptide (150 μg/ml) inhibits LSD-phage binding to theCXCR4-transfected 293T cells (P<0.03). The LSD peptide with LSD-phagewas co-incubated with CXCR4-transfected cells. Shown are the mean andstandard deviation from three separate experiments.

FIGS. 8A and 8B show targeting the tumor-associated lymphatics withhoming peptides linked to a pro-apoptotic peptide. The PPC1 orthotopicxenografted mice (10 mice/group) were systemically treated with 100μg/dose/mouse/biweekly of _(D)(KLAKLAK)₂-CREAGRKAC (SEQ ID NO:6),equimolar amounts of the uncoupled peptides, or with the vehicle (PBS).At termination, tumor weights were recorded, and frozen tissue sectionswere prepared for immunohistochemical analysis. The_(D)(KLAKLAK)₂-CREAGRKAC (SEQ ID NO:41) chimeric peptide greatly reducedthe number of tumor lymphatics (P<0.01) as determined from podoplaninstaining (A), whereas the blood vessel count (A; MECA-32 staining) andtumor volume (B) were unaffected.

FIGS. 9A and 9B show phage library screening for peptides homing tolymphatic vessels in C8161 xenograft tumors. FIG. 9A shows ex vivoselection. The CX7C phage library (5×10¹⁰ pfu) was incubated with 5×10⁷cells derived from C8161 xenograft tumors at 4° C. overnight. Lymphaticendothelial cells were isolated with anti-mouse podoplanin captured ontomagnetic beads. The phage that bound to lymphatic endothelial cells wererescued and amplified for subsequent screening. Three rounds of ex vivoselection yielded 250-fold enrichment of phage as compared to thebackground obtained with non-recombinant phage. FIG. 9B shows in vivoselection. The enriched phage pool from the third ex vivo selectionround was injected into the tail vein of a C8161 tumor mouse. Phage wererecovered from tumor tissue, amplified, and the selection was repeated.A 40-fold enrichment relative to non-recombinant phage was obtained intwo in vivo rounds.

FIG. 10A shows comparison of the homing of REA phage to orthotopic vs.subcutaneously xenografted PPC1 tumors in nude mice. The REA phage(5×10⁹ pfu) were injected into tumor mice by tail vein. Aftercirculation for 7 min, the bound phage were recovered from the tumorsand various control organs and titrated (P<0.03 for orthotopic versussubcutaneous tumors; n=3). FIG. 10B shows REA-phage binds to cellsuspensions derived from PPC1 tumors, but not to cultured PPC1 cells.

DETAILED DESCRIPTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a peptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the peptide are discussed, each and every combination andpermutation of peptide and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Disclosed herein are peptides that selectively target tumor lymphatics.In some aspects, the disclosed peptides can selectively targetlymphangiogenic vessels. In some aspects, the disclosed peptides canselectively bind markers present on tumor lymphatics. In some aspects,the disclosed peptides can selectively bind markers present on tumorsand tumor cells. In some aspects, the disclosed peptides can selectivelybind markers present on both tumors and tumor lymphatics. Thus, thedisclosed peptides can be used, for example, to deliver moieties, suchas therapeutic and/or detection moieties to tumor lymphatics, tumors,tumor cells and/or lymphangiogenic vessels. Also disclosed are methodsof, for example, selectively targeting tumor lymphatics in a subject,selectively targeting a tumor cell in a subject, and selectivelytargeting tumor lymphatics and tumor cells.

The metastatic spread of tumor cells is the underlying cause of mostcancer-related deaths. Clinical and pathological evidence confirms thatthe metastatic spread of tumors via lymphatic vessels to local/regionallymph nodes is an early event in metastatic disease for many solid humantumors. Tumors that secrete VEGF-C or VEGF-D induce lymphangiogenesis byactivating VEGFR-3 on lymphatic vessels, a process known as tumorlymphangiogenesis. Lymphangiogesis is the formation of lymphatic vesselsfrom pre-existing lymphatic vessels, in a method believed to be similarto blood vessel development or angiogenesis. Lymphangiogenesis alsoplays an important physiological role in homeostasis, metabolism andimmunity. Lymphatic vessel formation has also been implicated in anumber of pathological conditions including neoplasm metastasis, oedema,rheumatoid arthritis, psoriasis and impaired wound healing.

As used herein, a “targeting peptide” is peptide or polypeptide thatbinds to a target, such as a cell. For example, a targeting peptide candisplay selective targeting activity. The terms “selective targeting” or“selective homing” as used herein each refer to a preferentiallocalization of a compound or composition, such as the disclosedcompositions, that results in an amount of the compound or compositionin a target tissue that is, for example, about 2-fold greater than anamount of the peptide in a control tissue, about 5-fold or greater, orabout 10-fold or greater. For example, the terms “selective targeting”and “selective homing” can refer to binding or accumulation of acompound or composition, such as the disclosed compositions in a targettissue concomitant with an absence of targeting to a control tissue orthe absence of targeting to all control tissues.

A. Compositions

Disclosed herein are compositions and methods relating to novel peptidesand conjugates that selectively target tumor lymphatics.

1. Peptides

Disclosed herein are peptides that target, bind to and/or home to tumorlymphatics, such as lymphatic vessels in and around tumors (which canalso be referred to as tumor-associated lymphatic vessels), and/orlymphangiogenic vessels. The disclosed peptides preferably selectivelybind to tumor lymphatics. The disclosed peptides can have a variety ofstructures. For example, in some forms, the amino acid sequence of thedisclosed peptide can be CLSDGK (SEQ ID NO:2), CLSDGK (SEQ ID NO:2) withone, two or three conservative amino acid substitutions, CLSDGK (SEQ IDNO:2) with one non-conservative amino acid substitution, CLSDGK (SEQ IDNO:2) with one non-conservative amino acid substitution and one, two orthree conservative amino acid substitutions, or a fragment of any ofthese forms having at least four consecutive amino acids. Also providedis an isolated peptide, wherein the peptide comprises an amino acidsequence, wherein the amino acid sequence has at least 70% sequenceidentity with CLSDGK (SEQ ID NO:2).

As a further example, the amino acid sequence of the disclosed peptidecan be CLSDGKRKC (SEQ ID NO:4), CLSDGKRKC (SEQ ID NO:4) with one, two,three or four conservative amino acid substitutions, CLSDGKRKC (SEQ IDNO:4) with one, two or three non-conservative amino acid substitution,CLSDGKRKC (SEQ ID NO:4) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CLSDGKRKC (SEQ IDNO:4).

As a further example, the amino acid sequence of the disclosed peptidecan be CLSDGKPVS (SEQ ID NO:3), CLSDGKPVS (SEQ ID NO:3) with one, two,three or four conservative amino acid substitutions, CLSDGKPVS (SEQ IDNO:3) with one, two or three non-conservative amino acid substitution,CLSDGKPVS (SEQ ID NO:3) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CLSDGKPVS (SEQ IDNO:3).

As a further example, the amino acid sequence of the disclosed peptidecan be CASLSCR (SEQ ID NO:10), CASLSCR (SEQ ID NO:10) with one, two orthree conservative amino acid substitutions, CASLSCR (SEQ ID NO:10) withone or two non-conservative amino acid substitution, CASLSCR (SEQ IDNO:10) with one or two non-conservative amino acid substitution and one,two or three conservative amino acid substitutions, or a fragment of anyof these forms having at least four consecutive amino acids. Alsoprovided is an isolated peptide, wherein the peptide comprises an aminoacid sequence, wherein the amino acid sequence has at least 65% sequenceidentity with CASLSCR (SEQ ID NO:10).

As a further example, the amino acid sequence of the disclosed peptidecan be CLDGGRPKC (SEQ ID NO:5), CLDGGRPKC (SEQ ID NO:5) with one, two,three or four conservative amino acid substitutions, CLDGGRPKC (SEQ IDNO:5) with one, two or three non-conservative amino acid substitution,CLDGGRPKC (SEQ ID NO:5) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CLDGGRPKC (SEQ IDNO:5).

As a further example, the amino acid sequence of the disclosed peptidecan be CREAGRKAC (SEQ ID NO:6), CREAGRKAC (SEQ ID NO:6) with one, two,three or four conservative amino acid substitutions, CREAGRKAC (SEQ IDNO:6) with one, two or three non-conservative amino acid substitution,CREAGRKAC (SEQ ID NO:6) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CREAGRKAC (SEQ IDNO:6),

As a further example, the amino acid sequence of the disclosed peptidecan be CSMSAKKKC (SEQ ID NO:7), CSMSAKKKC (SEQ ID NO:7) with one, two,three or four conservative amino acid substitutions, CSMSAKKKC (SEQ IDNO:7) with one, two or three non-conservative amino acid substitution,CSMSAKKKC (SEQ ID NO:7) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CSMSAKKKC (SEQ IDNO:7).

As a further example, the amino acid sequence of the disclosed peptidecan be CKTRVSCGV (SEQ ID NO:8), CKTRVSCGV (SEQ ID NO:8) with one, two,three or four conservative amino acid substitutions, CKTRVSCGV (SEQ IDNO:8) with one, two or three non-conservative amino acid substitution,CKTRVSCGV (SEQ ID NO:8) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CKTRVSCGV (SEQ IDNO:8).

As a further example, the amino acid sequence of the disclosed peptidecan be CAGRRSAYC (SEQ ID NO:9), CAGRRSAYC (SEQ ID NO:9) with one, two,three or four conservative amino acid substitutions, CAGRRSAYC (SEQ IDNO:9) with one, two or three non-conservative amino acid substitution,CAGRRSAYC (SEQ ID NO:9) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CAGRRSAYC (SEQ IDNO:9).

As a further example, the amino acid sequence of the disclosed peptidecan be CSGGKVLDC (SEQ ID NO:11), CSGGKVLDC (SEQ ID NO:11) with one, two,three or four conservative amino acid substitutions, CSGGKVLDC (SEQ IDNO:11) with one, two or three non-conservative amino acid substitution,CSGGKVLDC (SEQ ID NO:11) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, whereinthe peptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CSGGKVLDC (SEQ IDNO:11).

As a further example, the peptide can comprise an amino acid sequence,where the amino acid sequence is XRTX (SEQ ID NO:59), where X is R or K(SEQ ID NOs:12-15). For example, the amino acid sequence of thedisclosed peptide can be CGNKRTRGC (SEQ ID NO:16), CGNKRTRGC (SEQ IDNO:16) with one, two, three or four conservative amino acidsubstitutions, CGNKRTRGC (SEQ ID NO:16) with one, two or threenon-conservative amino acid substitution, CGNKRTRGC (SEQ ID NO:16) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions, or a fragment ofany of these forms having at least four consecutive amino acids. Alsoprovided is an isolated peptide, where the peptide comprises an aminoacid sequence, wherein the amino acid sequence has at least 65% sequenceidentity with CGNKRTRGC (SEQ ID NO:16). In some aspects, the amino acidsequence of the disclosed peptide does not consist of CGNKRTRGC (SEQ IDNO:16).

As a further example, the amino acid sequence of the disclosed peptidecan be CNRRTKAGC (SEQ ID NO:17), CNRRTKAGC (SEQ ID NO:17) with one, two,three or four conservative amino acid substitutions, CNRRTKAGC (SEQ IDNO:17) with one, two or three non-conservative amino acid substitution,CNRRTKAGC (SEQ ID NO:17) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, where thepeptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CNRRTKAGC (SEQ IDNO:17). In some aspects, the amino acid sequence of the disclosedpeptide does not consist of CNRRTKAGC (SEQ ID NO:17).

As a further example, the amino acid sequence of the disclosed peptidecan be CNKRTRGGC (SEQ ID NO:18), CNKRTRGGC (SEQ ID NO:18) with one, two,three or four conservative amino acid substitutions, CNKRTRGGC (SEQ IDNO:18) with one, two or three non-conservative amino acid substitution,CNKRTRGGC (SEQ ID NO:18) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions, or a fragment of any of these forms having at least fourconsecutive amino acids. Also provided is an isolated peptide, where thepeptide comprises an amino acid sequence, wherein the amino acidsequence has at least 65% sequence identity with CNKRTRGGC (SEQ IDNO:18). In some aspects, the amino acid sequence of the disclosedpeptide does not consist of CNKRTRGGC (SEQ ID NO:18).

As a further example, the amino acid sequence of the disclosed peptidecan consist of CLSDGK (SEQ ID NO:2), CASLSCR (SEQ ID NO:10), CLDGGRPKC(SEQ ID NO:5), CREAGRKAC (SEQ ID NO:6), CSMSAKKKC (SEQ ID NO:7),CKTRVSCGV (SEQ ID NO:8), CAGRRSAYC (SEQ ID NO:9), CSGGKVLDC (SEQ IDNO:11), CGNKRTRGC (SEQ ID NO:16), CNRRTKAGC (SEQ ID NO:17), CNKRTRGGC(SEQ ID NO:18), CLSDGKRKC (SEQ ID NO:4), or CLSDGKPVS (SEQ ID NO:3).

The disclosed peptide can have any suitable length. For example, thepeptide can have a length of up to 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50amino acids. The disclosed polypeptides can be, for example, 4 to about50 amino acids in length. The disclosed polypeptides can be, forexample, less than about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39,38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 aminoacids in length.

For example, the disclosed peptide can have a length of from 4 to about10 amino acids, from 4 to about 15 amino acids, from 4 to about 20 aminoacids, from 4 to about 25 amino acids, from 4 to about 30 amino acids,from 4 to about 35 amino acids, from 4 to about 40 amino acids, from 4to about 45 amino acids, from 4 to about 50 amino acids. For example,the disclosed peptide can have a length of from 5 to about 10 aminoacids, from 5 to about 15 amino acids, from 5 to about 20 amino acids,from 5 to about 25 amino acids, from 5 to about 30 amino acids, from 5to about 35 amino acids, from 5 to about 40 amino acids, from 5 to about45 amino acids, from 5 to about 50 amino acids. For example, thedisclosed peptide can have a length of from 6 to about 10 amino acids,from 6 to about 15 amino acids, from 6 to about 20 amino acids, from 6to about 25 amino acids, from 6 to about 30 amino acids, from 6 to about35 amino acids, from 6 to about 40 amino acids, from 6 to about 45 aminoacids, from 6 to about 50 amino acids. For example, the disclosedpeptide can have a length of from 7 to about 10 amino acids, from 7 toabout 15 amino acids, from 7 to about 20 amino acids, from 7 to about 25amino acids, from 7 to about 30 amino acids, from 7 to about 35 aminoacids, from 7 to about 40 amino acids, from 7 to about 45 amino acids,from 7 to about 50 amino acids. For example, the disclosed peptide canhave a length of from 8 to about 10 amino acids, from 8 to about 15amino acids, from 8 to about 20 amino acids, from 8 to about 25 aminoacids, from 8 to about 30 amino acids, from 8 to about 35 amino acids,from 8 to about 40 amino acids, from 8 to about 45 amino acids, from 8to about 50 amino acids. For example, the disclosed peptide can have alength of from 9 to about 10 amino acids, from 9 to about 15 aminoacids, from 9 to about 20 amino acids, from 9 to about 25 amino acids,from 9 to about 30 amino acids, from 9 to about 35 amino acids, from 9to about 40 amino acids, from 9 to about 45 amino acids, from 9 to about50 amino acids.

The disclosed peptides can be artificial sequences and can besynthesized in vitro and/or recombinantly. The disclosed polypeptidescan be peptides that are not naturally occurring proteins and can bepeptides that have at least two contiguous sequences that are notcontiguous in a naturally occurring protein.

The disclosed peptides and compositions also can comprise anycombination of two, three, or more of the disclosed peptides or aminoacid sequences. Thus, disclosed are peptides comprising any one, two,three, or more of the herein disclosed peptides or amino acid sequences.The peptides can be combined in any suitable manner, including, forexample, as a single amino acid chain (that is a fusion of thepeptides), via linkers, via branched linkers, and attached individuallyor together to a structure. Also disclosed are bifunctional peptides,which contain one or more of the disclosed peptides fused to one or moresecond peptides having one or more separate functions. Such bifunctionalpeptides can have at least two functions conferred by different portionsof the full-length molecule and can, for example, display pro-apoptoticactivity in addition to the ability to target the tumor lymphatic.

Also disclosed are multivalent peptides that can include at least two ofthe disclosed peptides each independently containing one or more of thedisclosed amino acid sequences. The multivalent peptide can have, forexample, at least three, at least five or at least ten of such peptideseach independently containing a disclosed amino acid sequence. In someaspects, the multivalent peptide can have two, three, four, five, six,seven, eight, nine, ten, fifteen or twenty identical or non-identicalpeptides and/or amino acid sequences. In some aspects, the multivalentpeptide can contain identical peptides and/or amino acid sequences. Insome aspects, the multivalent peptide can contain contiguous identicalor non-identical peptides and/or amino acid sequences, which are or arenot separated by any intervening amino acids.

2. Conjugate

Also provided herein is a conjugate comprising any one or more of theherein disclosed peptides and one or more moieties. In general, themoiety can be a substance that acts upon the target cell(s) or tissue tobing about a desired effect. In some aspects, the disclosed conjugatecan target, bind to and/or home to tumor lymphatics, such as lymphaticvessels in and around tumors, and/or lymphangiogenic vessels. Thedisclosed peptides preferably selectively bind to tumor lymphatics.Thus, the effect can, for example, be the labeling, activating,repressing, or killing of the target cell(s) or tissue.

The moiety can be, for example, a therapeutic moiety or a detectablemoiety, a cytotoxic agent, an anti-lymphangiogenic agent, a cancerchemotherapeutic agent, a pro-apoptotic polypeptide, a graftedpolypeptide, a virus, a cell, or a liposome. Thus, the moiety can be asmall molecule, pharmaceutical drug, toxin, fatty acid, detectablemarker, conjugating tag, nanoparticle, or enzyme. For example, themoiety of the disclosed conjugate can be a pro-apoptotic peptide.Examples of pro-apopototic peptides are the amino acid sequence_(D)(KLAKLAK)₂ (SEQ ID NO:19), tumor necrosis factor (Curnis et al.,Cancer Res. 64, 565-71, 2004) and tachyplesin (Chen et al., Cancer res.61, 2434-8, 2001). Many other pro-apoptotic peptides and compounds areknown and can be used with and in the disclosed compositions, conjugatesand methods.

Examples of small molecules and pharmaceutical drugs that can beconjugated to a peptide are known in the art. The moiety can be acytotoxic small molecule or drug that kills the target cell. The smallmolecule or drug can be designed to act on any critical cellularfunction or pathway. For example, the small molecule or drug can inhibitthe cell cycle, activate protein degradation, induce apoptosis, modulatekinase activity, or modify cytoskeletal proteins. Any known or newlydiscovered cytotoxic small molecule or drugs is contemplated for usewith the peptides.

The moiety can be a toxin that kills the targeted cell. Non-limitingexamples of toxins include abrin, modeccin, ricin and diphtheria toxin.Other known or newly discovered toxins are contemplated for use with theprovided conjugates.

Fatty acids (i.e., lipids) that can be conjugated to the providedconjugates include those that allow the efficient incorporation of thepeptide into liposomes. Generally, the fatty acid is a polar lipid.Thus, the fatty acid can be a phospholipid. The provided conjugates cancomprise either natural or synthetic phospholipid. The phospholipids canbe selected from phospholipids containing saturated or unsaturated monoor disubstituted fatty acids and combinations thereof. Thesephospholipids can be dioleoylphosphatidylcholine,dioleoylphosphatidylserine, dioleoylphosphatidylethanolamine,dioleoylphosphatidylglycerol, dioleoylphosphatidic acid,palmitoyloleoylphosphatidylcholine, palmitoyloleoylphosphatidylserine,palmitoyloleoylphosphatidylethanolamine,palmitoyloleoylphophatidylglycerol, palmitoyloleoylphosphatidic acid,palmitelaidoyloleoylphosphatidylcholine,palmitelaidoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylethanolamine,palmitelaidoyloleoylphosphatidylglycerol,palmitelaidoyloleoylphosphatidic acid,myristoleoyloleoylphosphatidylcholine,myristoleoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylethanoamine,myristoleoyloleoylphosphatidylglycerol, myristoleoyloleoylphosphatidicacid, dilinoleoylphosphatidylcholine, dilinoleoylphosphatidylserine,dilinoleoylphosphatidylethanolamine, dilinoleoylphosphatidylglycerol,dilinoleoylphosphatidic acid, palmiticlinoleoylphosphatidylcholine,palmiticlinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylethanolamine,palmiticlinoleoylphosphatidylglycerol, palmiticlinoleoylphosphatidicacid. These phospholipids may also be the monoacylated derivatives ofphosphatidylcholine (lysophophatidylidylcholine), phosphatidylserine(lysophosphatidylserine), phosphatidylethanolamine(lysophosphatidylethanolamine), phophatidylglycerol(lysophosphatidylglycerol) and phosphatidic acid (lysophosphatidicacid). The monoacyl chain in these lysophosphatidyl derivatives may bepalimtoyl, oleoyl, palmitoleoyl, linoleoyl myristoyl or myristoleoyl.The phospholipids can also be synthetic. Synthetic phospholipids arereadily available commercially from various sources, such as AVANTIPolar Lipids (Albaster, Ala.); Sigma Chemical Company (St. Louis, Mo.).These synthetic compounds may be varied and may have variations in theirfatty acid side chains not found in naturally occurring phospholipids.The fatty acid can have unsaturated fatty acid side chains with C14,C16, C18 or C20 chains length in either or both the PS or PC. Syntheticphospholipids can have dioleoyl (18:1)-PS; palmitoyl (16:0)-oleoyl(18:1)-PS, dimyristoyl (14:0)-PS; dipalmitoleoyl (16:1)-PC, dipalmitoyl(16:0)-PC, dioleoyl (18:1)-PC, palmitoyl (16:0)-oleoyl (18:1)-PC, andmyristoyl (14:0)-oleoyl (18:1)-PC as constituents. Thus, as an example,the provided conjugates can comprise palmitoyl 16:0.

The moiety of the disclosed conjugate can be a detection moiety.Detectable moieties/markers include any substance that can be used tolabel or stain a target tissue or cell(s). Non-limiting examples ofdetectable markers include radioactive isotopes, enzymes, fluorophores,and quantum dots (Qdot®). For example, the detection moiety can be anenzyme, biotin, metal, or epitope tag. Other known or newly discovereddetectable markers are contemplated for use with the providedconjugates.

Fluorophores are compounds or molecules that luminesce. Typicallyfluorophores absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength. Representativefluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS;4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT);5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-Imethylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; AcidFuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs—AutoFluorescentProtein—(Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X;Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate;APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA;ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FLATP; Bodipy FI-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson-; Calcium Green; Calcium Green-1 Ca²⁺Dye;Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺;Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); CascadeBlue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (CyanFluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; CoumarinPhalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan;Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP;cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydrorhodamine123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline);FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline;Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; OregonGreen™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; PacificBlue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primuline;Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T;Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™(super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl) quinolinium); Stilbene;Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductornanoparticles such as quantum dots; or caged fluorophore (which can beactivated with light or other electromagnetic energy source), or acombination thereof.

The moiety can be a nanoparticle, such as a heat generating nanoshell.As used herein, “nanoshell” is a nanoparticle having a discretedielectric or semi-conducting core section surrounded by one or moreconducting shell layers. U.S. Pat. No. 6,530,944 is hereby incorporatedby reference herein in its entirety for its teaching of the methods ofmaking and using metal nanoshells. Nanoshells can be formed with a coreof a dielectric or inert material such as silicon, coated with amaterial such as a highly conductive metal which can be excited usingradiation such as near infrared light (approximately 800 to 1300 nm).Upon excitation, the nanoshells emit heat. The resulting hyperthermiacan kill the surrounding cell(s) or tissue. The combined diameter of theshell and core of the nanoshells ranges from the tens to the hundreds ofnanometers. Near infrared light is advantageous for its ability topenetrate tissue. Other types of radiation can also be used, dependingon the selection of the nanoparticle coating and targeted cells.Examples include x-rays, magnetic fields, electric fields, andultrasound. The particles can also be used to enhance imaging,especially using infrared diffuse photon imaging methods. Targetingmolecules can be antibodies or fragments thereof, ligands for specificreceptors, or other proteins specifically binding to the surface of thecells to be targeted.

The moiety can be covalently linked to the disclosed peptide. The moietycan be linked to the amino terminal end of the disclosed peptide. Themoiety can be linked to the carboxy terminal end of the disclosedpeptide. The moiety can be linked to an amino acid within the disclosedpeptide. The herein provided conjugates can further comprise a linkerconnecting the moiety and disclosed peptide. The disclosed peptide canalso be conjugated to a coating molecule such as bovine serum albumin(BSA) (see Tkachenko et al., (2003) J Am Chem Soc, 125, 4700-4701) thatcan be used to coat the Nanoshells with the peptide.

Protein crosslinkers that can be used to crosslink the moiety to thedisclosed peptide are known in the art and are defined based on utilityand structure and include DSS (Disuccinimidylsuberate), DSP(Dithiobis(succinimidylpropionate)), DTSSP (3,3′-Dithiobis(sulfosuccinimidylpropionate)), SULFO BSOCOES(Bis[2-(sulfosuccinimdooxycarbonyloxy) ethyl]sulfone), BSOCOES(Bis[2-(succinimdooxycarbonyloxy)ethyl]sulfone), SULFO DST(Disulfosuccinimdyltartrate), DST (Disuccinimdyltartrate), SULFO EGS(Ethylene glycolbis(succinimidylsuccinate)), EGS (Ethyleneglycolbis(sulfosuccinimidylsuccinate)), DPDPB(1,2-Di[3′-(2′-pyridyldithio) propionamido]butane), BSSS(Bis(sulfosuccinimdyl) suberate), SMPB(Succinimdyl-4-(p-maleimidophenyl) butyrate), SULFO SMPB(Sulfosuccinimdyl-4-(p-maleimidophenyl) butyrate), MBS(3-Maleimidobenzoyl-N-hydroxysuccinimide ester), SULFO MBS(3-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SIAB(N-Succinimidyl(4-iodoacetyl)aminobenzoate), SULFO SIAB(N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SMCC(Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SULFOSMCC (Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate),NHS LC SPDP (Succinimidyl-6-[3-(2-pyridyldithio) propionamido)hexanoate), SULFO NHS LC SPDP (Sulfosuccinimidyl-6-[3-(2-pyridyldithio)propionamido) hexanoate), SPDP (N-Succinimdyl-3-(2-pyridyldithio)propionate), NHS BROMOACETATE (N-Hydroxysuccinimidylbromoacetate), NHSIODOACETATE (N-Hydroxysuccinimidyliodoacetate), MPBH(4-(N-Maleimidophenyl) butyric acid hydrazide hydrochloride), MCCH(4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid hydrazidehydrochloride), MBH (m-Maleimidobenzoic acid hydrazidehydrochloride),SULFO EMCS(N-(epsilon-Maleimidocaproyloxy) sulfosuccinimide),EMCS(N-(epsilon-Maleimidocaproyloxy) succinimide), PMPI(N-(p-Maleimidophenyl) isocyanate), KMUH (N-(kappa-Maleimidoundecanoicacid) hydrazide), LC SMCC(Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy(6-amidocaproate)),SULFO GMBS (N-(gamma-Maleimidobutryloxy) sulfosuccinimide ester), SMPH(Succinimidyl-6-(beta-maleimidopropionamidohexanoate)), SULFO KMUS(N-(kappa-Maleimidoundecanoyloxy)sulfosuccinimide ester), GMBS(N-(gamma-Maleimidobutyrloxy) succinimide), DMP (Dimethylpimelimidatehydrochloride), DMS (Dimethylsuberimidate hydrochloride), MHBH (Wood'sReagent) (Methyl-p-hydroxybenzimidate hydrochloride, 98%), DMA(Dimethyladipimidate hydrochloride).

The moiety of the disclosed conjugate can be a cellular internalizationtransporter or sequence. The cellular internalization sequence can beany internalization sequence known or newly discovered in the art, orconservative variants thereof. Non-limiting examples of cellularinternalization transporters and sequences include Antennapediasequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC,Pep-1, SynB1, Pep-7, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, andBGTC (Bis-Guanidinium-Tren-Cholesterol) (see Table 1).

TABLE 1 Cell Internalization Transporters Name Sequence SEQ ID NO AntpRQPKIWFPNRRKPWKK (SEQ ID NO: 44) HIV-Tat GRKKRRQRPPQ (SEQ ID NO: 45)Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 46) Antp-3A RQIAIWFQNRRMKWAA(SEQ ID NO: 47) Tat RKKRRQRRR (SEQ ID NO: 48) Buforin IITRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 49) TransportanGWTLNSAGYLLGKINKALAALAKKIL (SEQ ID NO: 50) model amphipathicKLALKLALKALKAALKLA (SEQ ID NO: 51) peptide (MAP) K-FGF AAVALLPAVLLALLAP(SEQ ID NO: 52) Ku70 VPMLK-PMLKE (SEQ ID NO: 53) PrionMANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 54) pVEC LLIILRRRIRKQAHAHSK(SEQ ID NO: 55) Pep-1 KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 56) SynB1RGGRLSYSRRRFSTSTGR (SEQ ID NO: 57) Pep-7 SDLWEMMMVSLACQY (SEQ ID NO: 58)HN-1 TSPLNIHNGQKL (SEQ ID NO: 59) BGSC (Bis- Guanidinium- Spermidine-Cholesterol)

BGTC (Bis- Guanidinium-Tren- Cholesterol)

Thus, the provided polypeptide can further comprise the amino acidsequence SEQ ID NO:44, SEQ ID NO:45 (Bucci, M. et al. 2000. Nat. Med. 6,1362-1367), SEQ ID NO:46 (Derossi, D., et al. 1994. Biol. Chem. 269,10444-10450), SEQ ID NO:47 (Fischer, P. M. et al. 2000. J. Pept. Res.55, 163-172), SEQ ID NO:48 (Frankel, A. D. & Pabo, C. O. 1988. Cell 55,1189-1193; Green, M. & Loewenstein, P. M. 1988. Cell 55, 1179-1188), SEQID NO:49 (Park, C. B., et al. 2000. Proc. Natl. Acad. Sci. USA 97,8245-8250), SEQ ID NO:50 (Pooga, M., et al. 1998. FASEB J. 12, 67-77),SEQ ID NO:51 (Oehlke, J. et al. 1998. Biochim. Biophys. Acta. 1414,127-139), SEQ ID NO:52 (Lin, Y. Z., et al. 1995. J. Biol. Chem. 270,14255-14258), SEQ ID NO:53 (Sawada, M., et al. 2003. Nature Cell Biol.5, 352-357), SEQ ID NO:54 (Lundberg, P. et al. 2002. Biochem. Biophys.Res. Commun. 299, 85-90), SEQ ID NO:55 (Elmquist, A., et al. 2001. Exp.Cell Res. 269, 237-244), SEQ ID NO:56 (Morris, M. C., et al. 2001.Nature Biotechnol. 19, 1173-1176), SEQ ID NO:57 (Rousselle, C. et al.2000. Mol. Pharmacol. 57, 679-686), SEQ ID NO:58 (Gao, C. et al. 2002.Bioorg. Med. Chem. 10, 4057-4065), or SEQ ID NO:59 (Hong, F. D. &Clayman, G. L. 2000. Cancer Res. 60, 6551-6556). The providedpolypeptide can further comprise BGSC(Bis-Guanidinium-Spermidine-Cholesterol) or BGTC(Bis-Guanidinium-Tren-Cholesterol) (Vigneron, J. P. et al. 1998. Proc.Natl. Acad. Sci. USA. 93, 9682-9686). The preceding references arehereby incorporated herein by reference in their entirety for theteachings of cellular internalization vectors and sequences. Any otherinternalization sequences now known or later identified can be combinedwith a polypeptide disclosed herein.

3. Polypeptides and Peptides i. Protein Variants

Protein variants and derivatives are well understood by those of skillin the art and in can involve amino acid sequence modifications. Forexample, amino acid sequence modifications typically fall into one ormore of three classes: substitutional, insertional or deletionalvariants. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Insertions ordinarily will be smaller insertions than those ofamino or carboxyl terminal fusions, for example, on the order of one tofour residues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTable 2 and are referred to as conservative substitutions.

TABLE 2 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala Ser Arg Lys; Gln AsnGln; His Asp Glu Cys Ser Gln Asn, Lys Glu Asp Gly Pro His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table2, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

Specifically disclosed are variants of these and other polypeptidesherein disclosed which have at least, 65%, 70% or 75% or 80% or 85% or90% or 95% homology to the stated sequence. Those of skill in the artreadily understand how to determine the homology of two proteins. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed peptides. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent than the amino acids shown in Table 2. Theopposite stereo isomers of naturally occurring peptides are disclosed,as well as the stereo isomers of peptide analogs. These amino acids canreadily be incorporated into polypeptide chains by charging tRNAmolecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEngineering Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are herein)incorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂—CH₂ (cis andtrans), —COCH₂—CH(OH)CH₂—, and —CHH₂SO— (These and others can be foundin Spatola, A. F. in Chemistry and Biochemistry of Amino Acids,Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,Peptide Backbone Modifications (general review); Morley, Trends PharmSci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. Life Sci38:1243-1249 (1986) (—CHH₂—S); Hann J. Chem. Soc Perkin Trans. 1307-314(1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

4. Nucleic Acid

Also provided herein is an isolated nucleic acid encoding any of theherein disclosed peptides. Also provided herein is an isolated nucleicacid encoding any of the herein disclosed peptides further comprising anucleic acid encoding a internalization sequence. For example, thecellular internalization can comprise an amino acid sequence of aprotein selected from a group consisting of Antennapedia, TAT, HIV-Tat,Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (modelamphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7,HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol and BGTC(Bis-Guanidinium-Tren-Cholesterol.

i. Nucleotides and Related Molecules

The disclosed nucleic acids can be made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell, that the expressedmRNA will typically be made up of A, C, G, and U. Likewise, it isunderstood that if, for example, an antisense molecule is introducedinto a cell or cell environment through for example exogenous delivery,it is advantageous that the antisense molecule be made up of nucleotideanalogs that reduce the degradation of the antisense molecule in thecellular environment.

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate). There are manyvarieties of these types of molecules available in the art and availableherein.

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties. There are many varieties of these typesof molecules available in the art and available herein.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid. There are many varieties of these types of molecules available inthe art and available herein.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of thesetypes of molecules available in the art and available herein.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

5. Cell Delivery Systems

Also provided is a vector comprising a nucleic acid encoding apolypeptide disclosed herein, wherein the nucleic acid is operablylinked to an expression control sequence. There are a number ofcompositions and methods which can be used to deliver nucleic acids tocells, either in vitro or in vivo. These methods and compositions canlargely be broken down into two classes: viral based delivery systemsand non-viral based delivery systems. For example, the nucleic acids canbe delivered through a number of direct delivery systems such as,electroporation, lipofection, calcium phosphate precipitation, plasmids,viral vectors, viral nucleic acids, phage nucleic acids, phages,cosmids, or via transfer of genetic material in cells or carriers suchas cationic liposomes. Appropriate means for transfection, includingviral vectors, chemical transfectants, or physico-mechanical methodssuch as electroporation and direct diffusion of DNA, are described by,for example, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991). Such methods are well knownin the art and readily adaptable for use with the compositions andmethods described herein. In certain cases, the methods will be modifiedto specifically function with large DNA molecules. Further, thesemethods can be used to target certain diseases and cell populations byusing the targeting characteristics of the carrier.

i. Nucleic Acid Based Delivery Systems

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acid into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. Viral vectors include, for example, Adenovirus,Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDSvirus, neuronal trophic virus, Sindbis and other RNA viruses; includingthese viruses with the HIV backbone. Also disclosed are any viralfamilies which share the properties of these viruses which make themsuitable for use as vectors. Retroviruses include Murine MaloneyLeukemia virus, MMLV, and retroviruses that express the desirableproperties of MMLV as a vector. Retroviral vectors are able to carry alarger genetic payload, i.e., a transgene or marker gene, than otherviral vectors, and for this reason are a commonly used vector. However,they are not as useful in non-proliferating cells. Adenovirus vectorsare relatively stable and easy to work with, have high titers, and canbe delivered in aerosol formulation, and can transfect non-dividingcells. Pox viral vectors are large and have several sites for insertinggenes, they are thermostable and can be stored at room temperature.Disclosed is a viral vector that has been engineered to suppress theimmune response of the host organism, elicited by the viral antigens.Example vectors of this type can carry coding regions for Interleukin 8or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

a. Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

b. Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virions are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

c. Adeno-Associated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorporated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and cancontain upstream elements and response elements.

d. Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

ii. Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Regarding liposomes, see,e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989);Feigner et al. Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987); U.S. Pat.No. 4,897,355. Furthermore, the compound can be administered as acomponent of a microcapsule that can be targeted to specific cell types,such as macrophages, or where the diffusion of the compound or deliveryof the compound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials can be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). In general, receptors areinvolved in pathways of endocytosis, either constitutive or ligandinduced. These receptors cluster in clathrin-coated pits, enter the cellvia clathrin-coated vesicles, pass through an acidified endosome inwhich the receptors are sorted, and then either recycle to the cellsurface, become stored intracellularly, or are degraded in lysosomes.The internalization pathways serve a variety of functions, such asnutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

iii. In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject's cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

iv. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and can contain upstream elementsand response elements.

a. Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells can be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell. Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293(1984)). They are usually between 10 and 300 by in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein and insulin), typically, one will usean enhancer from a eukaryotic cell virus for general expression.Preferred examples are the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

The promotor and/or enhancer can be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) can also contain sequencesnecessary for the termination of transcription which can affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b. Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker can be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

6. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically have at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99 percent homology to the stated sequence or the nativesequence. Those of skill in the art readily understand how to determinethe homology of two proteins or nucleic acids, such as genes. Forexample, the homology can be calculated after aligning the two sequencesso that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison can beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

7. Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization caninvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 8), 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

8. Antibodies

Also disclosed herein are antibodies that specifically bind any of thepeptides or polypeptides disclosed herein. The term “antibodies” is usedherein in a broad sense and includes both polyclonal and monoclonalantibodies. In addition to intact immunoglobulin molecules, alsoincluded in the term “antibodies” are fragments or polymers of thoseimmunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules or fragments thereof, as long as they arechosen for their ability to interact with a polypeptides disclosedherein.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces mono clonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes can be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment can be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies can also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

9. Pharmaceutical Carriers

The disclosed compositions can be used therapeutically in combinationwith a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material can be administered to a subject, alongwith the nucleic acid or vector, without causing any undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the pharmaceutical composition in which it iscontained. The carrier would naturally be selected to minimize anydegradation of the active ingredient and to minimize any adverse sideeffects in the subject, as would be well known to one of skill in theart.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions can include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions can also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration can include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions can be administered as a pharmaceuticallyacceptable acid- or base-addition salt, formed by reaction withinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid,and organic acids such as formic acid, acetic acid, propionic acid,glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,succinic acid, maleic acid, and fumaric acid, or by reaction with aninorganic base such as sodium hydroxide, ammonium hydroxide, potassiumhydroxide, and organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

The materials can be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These can be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

B. Methods 1. Targeting

Provided herein is a method of targeting one or more moieties to tumorlymphatics in a subject. The method can involve administering to thesubject a conjugate comprising any one or more of the herein disclosedpeptides and the one or more moieties. The one or more moieties can bedetection moieties, such as those disclosed herein. Thus, the method canfurther comprise detecting the tumor in the subject by detecting thepresence of the conjugate in the lymphatics of the subject.

The one or more moieties can be therapeutic moieties, such as thosedisclosed herein. Thus, wherein the subject has cancer, targeting of themoiety to the tumor lymphatics of the subject can inhibitlymphangiogenesis in the tumor in the subject.

2. Detecting

Also provided is a method of detecting cancer. The method can involvecontacting a biological sample with a conjugate comprising any one ormore of the herein disclosed peptides and one or more moieties, anddetecting the presence of the conjugate in lymphatics of the sample. Insome aspects of the disclosed method, detecting the presence of more ofthe one or more conjugate(s) in the lymphatics than a reference orcontrol amount indicates the presence of cancer.

3. Treating

Also provided is a method of treating cancer in a subject. The methodcan involve administering to the subject a conjugate comprising any oneor more of the herein disclosed peptides and one or more moieties. Theone or more moieties can be therapeutic moieties. Thus, in some aspects,the conjugate inhibits lymphangiogenesis in a tumor in the subject.conjugate induces apoptosis of the tumor lymphatics. In some aspects,the conjugate induces apoptosis of the tumor.

The cancer of the disclosed method can be breast cancer. Thus, thepolypeptide of the disclosed method can comprise the amino acid sequenceXRTX (SEQ ID NO:59), where X is R or K (SEQ ID NOs:12-15); CGNKRTRGC(SEQ ID NO:16), CGNKRTRGC (SEQ ID NO:16) with one, two, three or fourconservative amino acid substitutions, CGNKRTRGC (SEQ ID NO:16) withone, two or three non-conservative amino acid substitution, CGNKRTRGC(SEQ ID NO:16) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions; CNKRTRGGC (SEQ ID NO:18), CNKRTRGGC (SEQ ID NO:18) withone, two, three or four conservative amino acid substitutions, CNKRTRGGC(SEQ ID NO:18) with one, two or three non-conservative amino acidsubstitution, or CNKRTRGGC (SEQ ID NO:18) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions.

The cancer of the disclosed method can be cervical cancer. Thus, thepolypeptide of the disclosed method can comprise the amino acid sequenceXRTX (SEQ ID NO:59), where X is R or K (SEQ ID NOs:12-15); CNRRTKAGC(SEQ ID NO:17), CNRRTKAGC (SEQ ID NO:17) with one, two, three or fourconservative amino acid substitutions, CNRRTKAGC (SEQ ID NO:17) withone, two or three non-conservative amino acid substitution, or CNRRTKAGC(SEQ ID NO:17) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions.

The cancer of the disclosed method can be skin cancer. Thus, thepolypeptide of the disclosed method can comprise the amino acid sequenceCLSDGK (SEQ ID NO:2), CLSDGK (SEQ ID NO:2) with one, two or threeconservative amino acid substitutions, CLSDGK (SEQ ID NO:2) with onenon-conservative amino acid substitution, CLSDGK (SEQ ID NO:2) with onenon-conservative amino acid substitution and one, two or threeconservative amino acid substitutions; CLSDGKRKC (SEQ ID NO:4),CLSDGKRKC (SEQ ID NO:4) with one, two, three or four conservative aminoacid substitutions, CLSDGKRKC (SEQ ID NO:4) with one, two or threenon-conservative amino acid substitution, CLSDGKRKC (SEQ ID NO:4) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CLSDGKPVS (SEQ IDNO:3), CLSDGKPVS (SEQ ID NO:3) with one, two, three or four conservativeamino acid substitutions, CLSDGKPVS (SEQ ID NO:3) with one, two or threenon-conservative amino acid substitution, CLSDGKPVS (SEQ ID NO:3) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CLDGGRPKC (SEQ IDNO:5), CLDGGRPKC (SEQ ID NO:5) with one, two, three or four conservativeamino acid substitutions, CLDGGRPKC (SEQ ID NO:5) with one, two or threenon-conservative amino acid substitution, or CLDGGRPKC (SEQ ID NO:5)with one, two or three non-conservative amino acid substitution and one,two, three or four conservative amino acid substitutions.

The cancer of the disclosed method can be prostate cancer. Thus, thepolypeptide of the disclosed method can comprise the amino acid sequenceCREAGRKAC (SEQ ID NO:6), CREAGRKAC (SEQ ID NO:6) with one, two, three orfour conservative amino acid substitutions, CREAGRKAC (SEQ ID NO:6) withone, two or three non-conservative amino acid substitution, CREAGRKAC(SEQ ID NO:6) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions; CSMSAKKKC (SEQ ID NO:7), CSMSAKKKC (SEQ ID NO:7) withone, two, three or four conservative amino acid substitutions, CSMSAKKKC(SEQ ID NO:7) with one, two or three non-conservative amino acidsubstitution, CSMSAKKKC (SEQ ID NO:7) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions; CKTRVSCGV (SEQ ID NO:8),CKTRVSCGV (SEQ ID NO:8) with one, two, three or four conservative aminoacid substitutions, CKTRVSCGV (SEQ ID NO:8) with one, two or threenon-conservative amino acid substitution, CKTRVSCGV (SEQ ID NO:8) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CAGRRSAYC (SEQ IDNO:9), CAGRRSAYC (SEQ ID NO:9) with one, two, three or four conservativeamino acid substitutions, CAGRRSAYC (SEQ ID NO:9) with one, two or threenon-conservative amino acid substitution, CAGRRSAYC (SEQ ID NO:9) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CASLSCR (SEQ IDNO:10), CASLSCR (SEQ ID NO:10) with one, two or three conservative aminoacid substitutions, CASLSCR (SEQ ID NO:10) with one or twonon-conservative amino acid substitution, CASLSCR (SEQ ID NO:10) withone or two non-conservative amino acid substitution and one, two orthree conservative amino acid substitutions; CSGGKVLDC (SEQ ID NO:11),CSGGKVLDC (SEQ ID NO:11) with one, two, three or four conservative aminoacid substitutions, CSGGKVLDC (SEQ ID NO:11) with one, two or threenon-conservative amino acid substitution, or CSGGKVLDC (SEQ ID NO:11)with one, two or three non-conservative amino acid substitution and one,two, three or four conservative amino acid substitutions.

The cancer of the disclosed method can be pre-malignant prostate cancer.Thus, the polypeptide of the disclosed method can comprise the aminoacid sequence CAGRRSAYC (SEQ ID NO:9), CAGRRSAYC (SEQ ID NO:9) with one,two, three or four conservative amino acid substitutions, CAGRRSAYC (SEQID NO:9) with one, two or three non-conservative amino acidsubstitution, CAGRRSAYC (SEQ ID NO:9) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions; CASLSCR (SEQ ID NO:10), CASLSCR(SEQ ID NO:10) with one, two or three conservative amino acidsubstitutions, CASLSCR (SEQ ID NO:10) with one or two non-conservativeamino acid substitution, CASLSCR (SEQ ID NO:10) with one or twonon-conservative amino acid substitution and one, two or threeconservative amino acid substitutions; CSGGKVLDC (SEQ ID NO:11),CSGGKVLDC (SEQ ID NO:11) with one, two, three or four conservative aminoacid substitutions, CSGGKVLDC (SEQ ID NO:11) with one, two or threenon-conservative amino acid substitution, or CSGGKVLDC (SEQ ID NO:11)with one, two or three non-conservative amino acid substitution and one,two, three or four conservative amino acid substitutions.

The cancer of the disclosed method can be malignant prostate cancer.Thus, the polypeptide of the disclosed method can comprise the aminoacid sequence CREAGRKAC (SEQ ID NO:6), CREAGRKAC (SEQ ID NO:6) with one,two, three or four conservative amino acid substitutions, CREAGRKAC (SEQID NO:6) with one, two or three non-conservative amino acidsubstitution, CREAGRKAC (SEQ ID NO:6) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions; CSMSAKKKC (SEQ ID NO:7),CSMSAKKKC (SEQ ID NO:7) with one, two, three or four conservative aminoacid substitutions, CSMSAKKKC (SEQ ID NO:7) with one, two or threenon-conservative amino acid substitution, CSMSAKKKC (SEQ ID NO:7) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CKTRVSCGV (SEQ IDNO:8), CKTRVSCGV (SEQ ID NO:8) with one, two, three or four conservativeamino acid substitutions, CKTRVSCGV (SEQ ID NO:8) with one, two or threenon-conservative amino acid substitution, CKTRVSCGV (SEQ ID NO:8) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions.

The disclosed conjugates can be used to treat any disease whereuncontrolled cellular proliferation occurs such as cancers. Anon-limiting list of different types of cancers can be as follows:lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomasof solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas,gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas,histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas,AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers ingeneral.

A representative but non-limiting list of cancers that the disclosedcompositions can be used to treat is the following: lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloidleukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinoma of head and neck, kidney cancer,lung cancers such as small cell lung cancer and non-small cell lungcancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer,prostate cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, and epithelialcancer, renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, large bowel cancer, hematopoieticcancers; testicular cancer; colon and rectal cancers, prostatic cancer,or pancreatic cancer.

4. Diagnosing

Also provided is a method of determining normal, pre-malignant andmalignant prostate conditions in a subject. The method can involvecontacting a biological sample from the subject with a conjugatedisclosed herein, wherein the polypeptide comprises the amino acidsequence CREAGRKAC (SEQ ID NO:6), CREAGRKAC (SEQ ID NO:6) with one, two,three or four conservative amino acid substitutions, CREAGRKAC (SEQ IDNO:6) with one, two or three non-conservative amino acid substitution,CREAGRKAC (SEQ ID NO:6) with one, two or three non-conservative aminoacid substitution and one, two, three or four conservative amino acidsubstitutions; CSMSAKKKC (SEQ ID NO:7), CSMSAKKKC (SEQ ID NO:7) withone, two, three or four conservative amino acid substitutions, CSMSAKKKC(SEQ ID NO:7) with one, two or three non-conservative amino acidsubstitution, CSMSAKKKC (SEQ ID NO:7) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions; CKTRVSCGV (SEQ ID NO:8),CKTRVSCGV (SEQ ID NO:8) with one, two, three or four conservative aminoacid substitutions, CKTRVSCGV (SEQ ID NO:8) with one, two or threenon-conservative amino acid substitution, CKTRVSCGV (SEQ ID NO:8) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CAGRRSAYC (SEQ IDNO:9), CAGRRSAYC (SEQ ID NO:9) with one, two, three or four conservativeamino acid substitutions, CAGRRSAYC (SEQ ID NO:9) with one, two or threenon-conservative amino acid substitution, CAGRRSAYC (SEQ ID NO:9) withone, two or three non-conservative amino acid substitution and one, two,three or four conservative amino acid substitutions; CASLSCR (SEQ IDNO:10), CASLSCR (SEQ ID NO:10) with one, two or three conservative aminoacid substitutions, CASLSCR (SEQ ID NO:10) with one or twonon-conservative amino acid substitution, CASLSCR (SEQ ID NO:10) withone or two non-conservative amino acid substitution and one, two orthree conservative amino acid substitutions; CSGGKVLDC (SEQ ID NO:11),CSGGKVLDC (SEQ ID NO:11) with one, two, three or four conservative aminoacid substitutions, CSGGKVLDC (SEQ ID NO:11) with one, two or threenon-conservative amino acid substitution, or CSGGKVLDC (SEQ ID NO:11)with one, two or three non-conservative amino acid substitution and one,two, three or four conservative amino acid substitutions.

In some aspects of the disclosed method, selective binding of CAGRRSAYC(SEQ ID NO:9), CAGRRSAYC (SEQ ID NO:9) with one, two, three or fourconservative amino acid substitutions, CAGRRSAYC (SEQ ID NO:9) with one,two or three non-conservative amino acid substitution, CAGRRSAYC (SEQ IDNO:9) with one, two or three non-conservative amino acid substitutionand one, two, three or four conservative amino acid substitutions;CASLSCR (SEQ ID NO:10), CASLSCR (SEQ ID NO:10) with one, two or threeconservative amino acid substitutions, CASLSCR (SEQ ID NO:10) with oneor two non-conservative amino acid substitution, CASLSCR (SEQ ID NO:10)with one or two non-conservative amino acid substitution and one, two orthree conservative amino acid substitutions; or CSGGKVLDC (SEQ IDNO:11), CSGGKVLDC (SEQ ID NO:11) with one, two, three or fourconservative amino acid substitutions, CSGGKVLDC (SEQ ID NO:11) withone, two or three non-conservative amino acid substitution, or CSGGKVLDC(SEQ ID NO:11) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions is an indication of a pre-malignant prostate condition.

In some aspects of the disclosed method, selective binding of CREAGRKAC(SEQ ID NO:6), CREAGRKAC (SEQ ID NO:6) with one, two, three or fourconservative amino acid substitutions, CREAGRKAC (SEQ ID NO:6) with one,two or three non-conservative amino acid substitution, CREAGRKAC (SEQ IDNO:6) with one, two or three non-conservative amino acid substitutionand one, two, three or four conservative amino acid substitutions;CSMSAKKKC (SEQ ID NO:7), CSMSAKKKC (SEQ ID NO:7) with one, two, three orfour conservative amino acid substitutions, CSMSAKKKC (SEQ ID NO:7) withone, two or three non-conservative amino acid substitution, CSMSAKKKC(SEQ ID NO:7) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions; or CKTRVSCGV (SEQ ID NO:8), CKTRVSCGV (SEQ ID NO:8) withone, two, three or four conservative amino acid substitutions, CKTRVSCGV(SEQ ID NO:8) with one, two or three non-conservative amino acidsubstitution, CKTRVSCGV (SEQ ID NO:8) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions is an indication of a malignantprostate condition.

In some aspects of the disclosed method, a lack of selective binding ofCAGRRSAYC (SEQ ID NO:9), CAGRRSAYC (SEQ ID NO:9) with one, two, three orfour conservative amino acid substitutions, CAGRRSAYC (SEQ ID NO:9) withone, two or three non-conservative amino acid substitution, CAGRRSAYC(SEQ ID NO:9) with one, two or three non-conservative amino acidsubstitution and one, two, three or four conservative amino acidsubstitutions; CASLSCR (SEQ ID NO:10), CASLSCR (SEQ ID NO:10) with one,two or three conservative amino acid substitutions, CASLSCR (SEQ IDNO:10) with one or two non-conservative amino acid substitution, CASLSCR(SEQ ID NO:10) with one or two non-conservative amino acid substitutionand one, two or three conservative amino acid substitutions; orCSGGKVLDC (SEQ ID NO:8), CREAGRKAC (SEQ ID NO:3), CSMSAKKKC (SEQ IDNO:4), or CSGGKVLDC (SEQ ID NO:11), CSGGKVLDC (SEQ ID NO:11) with one,two, three or four conservative amino acid substitutions, CSGGKVLDC (SEQID NO:11) with one, two or three non-conservative amino acidsubstitution, or CSGGKVLDC (SEQ ID NO:11) with one, two or threenon-conservative amino acid substitution and one, two, three or fourconservative amino acid substitutions, is an indication of a normalprostate condition.

5. Screening

Also provided is a method of identifying an agent that targets tumorlymphatics. The method can involve, for example,

-   -   (a) contacting non-cancerous tissue with a library of candidate        agents under conditions sufficient to allow for selective        binding of agents to the non-cancerous tissue,    -   (b) collecting candidate agents that do not bind non-cancerous        tissue from step (a),    -   (c) contacting cancerous or pre-malignant tissue with the        candidate agents collected in step (b) under conditions        sufficient to allow for selective binding of agents to the        cancerous or pre-malignant tissue, and    -   (d) collecting candidate agents bound to lymphatic endothelial        cells from the cancerous or pre-malignant tissue, wherein        binding of candidate agents to lymphatic endothelial cells to        the cancerous or pre-malignant tissue identifies the candidate        agent as an agent that targets tumor lymphatics.

For example, the library of candidate agents can be from a phagelibrary.

The disclosed method can further comprise producing the candidate agentidentified as an agent that targets tumor lymphatics.

6. Administration

A composition disclosed herein, such as the disclosed peptides andconjugates, may be administered in a number of ways depending on whetherlocal or systemic treatment is desired, and on the area to be treated.For example, the compositions may be administered orally, parenterally(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscularinjection), by inhalation, extracorporeally, topically (includingtransdermally, ophthalmically, vaginally, rectally, intranasally) or thelike.

As used herein, “topical intranasal administration” means delivery ofthe compositions into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the nucleic acid or vector.Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. Thus, it is not possible to specify an exact amount for everycomposition. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein. Thus, effective dosages and schedules foradministering the compositions may be determined empirically, and makingsuch determinations is within the skill in the art. Useful dosage rangesfor the administration of the compositions are those large enough toproduce the desired effect. The dosage should not be so large as tocause adverse side effects, such as unwanted cross-reactions,anaphylactic reactions, and the like. Generally, the dosage will varywith the age, condition, sex and extent of the disease in the patient,route of administration, or whether other drugs are included in theregimen, and can be determined by one of skill in the art. The dosagecan be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or moredose administrations daily, for one or several days. Guidance can befound in the literature for appropriate dosages for given classes ofpharmaceutical products.

For example, a typical daily dosage of the disclosed peptides used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above.

Following administration of a disclosed composition, the efficacy of atherapeutic moiety can be assessed in various ways well known to theskilled practitioner. For instance, one of ordinary skill in the artwill understand that a composition disclosed herein is efficacious intreating or inhibiting cancer in a subject by observing that thecomposition reduces tumor growth or prevents a further increase inlymphangiogenesis.

C. Kits

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for producingconjugates, such as those disclosed herein, the kit comprising a peptideand a means for linking the peptide to a moiety. The kits also cancontain protocols for preparing the conjugate.

D. Uses

The disclosed compositions can be used in a variety of ways as researchtools. Other uses are disclosed, apparent from the disclosure, and/orwill be understood by those in the art.

E. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

1. Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

2. Peptide Synthesis

One method of producing the disclosed proteins is to link two or morepeptides or polypeptides together by protein chemistry techniques. Forexample, peptides or polypeptides can be chemically synthesized usingcurrently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to the disclosed proteins, for example, can be synthesizedby standard chemical reactions. For example, a peptide or polypeptidecan be synthesized and not cleaved from its synthesis resin whereas theother fragment of a peptide or protein can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof. (Grant G A (1992) SyntheticPeptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky Mand Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides may belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide-thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, Metal. Science,256:221 (1992)). This technique has been used to synthesize analogs ofprotein domains as well as large amounts of relatively pure proteinswith full biological activity (deLisle Milton R C et al., Techniques inProtein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

F. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of such peptides, reference to “thepeptide” is a reference to one or more peptides and equivalents thereofknown to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range is,expressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

The terms “targeting” or “homing”, as used herein can refer to thepreferential movement, binding and/or accumulation of a targetedcompound or composition, such as the disclosed compositions, at a siteor a location as compared to a non-targeted compound or composition. Forexample, in the context of in vivo administration to a subject,“targeting” or “homing” can refer to the preferential movement, binding,and/or accumulation of a compound or composition, such as the disclosedcompositions, in or at, for example, target tissue, target cells, and/ortarget structures as compared to non-target tissue, cells and/orstructures.

The term “target tissue” as used herein refers to an intended site foraccumulation of a targeted compound or composition, such as thedisclosed compositions, following administration to a subject. Forexample, the methods of the presently disclosed subject matter employ atarget tissue comprising endometriosis.

As used herein, “subject” includes, but is not limited to, animals,plants, bacteria, viruses, parasites and any other organism or entitythat has nucleic acid. The subject may be a vertebrate, morespecifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a birdor a reptile or an amphibian. The subject may to an invertebrate, morespecifically an arthropod (e.g., insects and crustaceans). The term doesnot denote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Apatient refers to a subject afflicted with a disease or disorder. Theterm “patient” includes human and veterinary subjects. In the context ofendometriosis and endometriosis cells, it is understood that a subjectis a subject that has or can have endometriosis and/or endometriosiscells.

By “treatment” is meant the medical management of a patient with theintent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

G. Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1 i. Experimental Procedures

Cell Lines, Mice, and Tumors:

The following cell lines were maintained in DMEM supplemented with 10%FCS: C8161 human melanoma, MDA-MB-435 human breast cancer, KRIB humanosteosarcoma, and human prostate cancer cells PPC1, DU145. LNCaP humanprostate cancer cell line was grown in RPMI 1640 medium with 10 mMHEPES, 1 mM sodium pyruvate and 1.5 g/L sodium bicarbonate supplementedwith 10% FCS. M12 human prostate cancer cell line was cultured in RPMI1640 with 5 μg/ml insulin-transferrin-sodium selenite (ITS), 2.5 μg/mlfungizone, 50 μg/ml gentamycin, 0.2 μM dexamethasone, 10 ng/ml epidermalgrowth factor (EGF), and 5% FCS (Bae et al., 1998). To produce tumors,nude BALB/c and C56BL/6 mice were subcutaneously (C8161, KRIB, and PPC1)or orthotopically (MDA-MB-453, PPC1, DU145, M12, and LNCaP) injectedwith 1×106 tumor cells. Transgenic mouse tumor models included TRAMPprostate cancer, MMTV-PyMT breast cancer, and K14-HPV16 cervical cancer.To initiate cervical carcinogenesis, female K14-HPV16 mice (Arbeit etal., 1994) were treated with 17β-estradiol (E2; (Arbeit et al., 1996;Giraudo et al., 2004). Briefly, one-month-old virgin female transgenic(heterozygous K14-HPV16, 1203#1) and nontransgenic (FVB/n) mice wereanesthetized with isoflurane, and continuous release pellets thatdeliver E2 at doses of 0.05 mg over 60 days (Innovative Research ofAmerica, Sarasota, Fla., USA) were implanted subcutaneously in thedorsal back skin. Subsequent pellets were implanted at 3 and 5 months ofage for a total of 6 months of hormone treatment. K14-HPV16 mice weremaintained in the FVB/n background (FVB/n; The Jackson Laboratory). Themice were maintained in accordance with the University of California,San Francisco (UCSF) institutional guidelines governing the care oflaboratory mice. The animal experimentation was approved by AnimalResearch Committees at UCSF or The Burnham Institute.

Phage Library and Screening:

An NNK-encoded CX7C library display on T7Select415-1 phage (Novagen) wasprepared as previously described (Laakkonen et al., 2002). Phageselection and validation have been described (Hoffman, 2004). A two-stepprocedure was designed for the selection of peptides targeting the tumorlymphatic vessels of pre-malignant prostate lesions and prostate tumor.First, the phage library was incubated with cells derived from normalprostate to subtract the phage that bind to normal prostate. Second, theanti-podoplanin magnetic beads were used to isolate lymphaticendothelial cells. 2-3 rounds of ex vivo selection and 2-3 rounds of invivo selections were performed. For the ex vivo selections, cellsuspensions were prepared from normal prostates of tumor-freelittermates of TRAMP mice, pre-malignant prostates of 14- to 16-week-oldTRAMP mice, and tumor tissues of 25- to 28-week-old TRAMP mice.Collagenase IA (1 mg/ml, Sigma) was used to disperse the tissues. About1×10⁷ normal prostate cells were incubated at 4° C. for 3 hrs with5×1010 plaque forming units (pfu) of T7 phage displaying a CX7C peptidelibrary. The samples were centrifuged at 1200 rpm for 10 min, thesupernatant (the normal prostate-subtracted phage library) was recoveredand then incubated overnight at 4° C. with 5×10⁷ cells derived frompre-malignant prostate tissue or prostate tumor. The cells were washedto remove unbound phage, incubated with rat anti-mouse podoplanin for 45min at 4° C., and washed three times with cold PBS containing 0.5% BSA.Podoplanin-positive cells were then isolated using M450 sheep anti-ratIgG Dynabeads (M450; Dynal, Oslo, Norway). Phage that bound to thepodoplanin-positive cell population were rescued and amplified in E.coli. In vivo phage library screening was performed as described(Laakkonen et al., 2002).

Homing Specificity of Phage:

In vivo homing specificity of phage was tested as described (Hoffman,2004). Briefly, mice bearing tumors were anesthetized and intravenouslyinjected with 5×10⁹ pfu of phage. After 7 min, the mice were perfusedthrough the heart with PBS containing 0.5% BSA. The tumor and controlorgans were dissected from each mouse and the phage were rescued andtittered. For histology analysis, the mice were perfused with 4% PFA 30min after the injection of phage. Tissues were embedded in Tissue-TekO.C.T. and 5 μm sections were prepared for phage immunostaining.

Antibodies and Immunohistology:

Custom immunization to produce a rabbit antiserum against mouse Prox-1was performed by Proteintech Inc. New Zealand White rabbits wereimmunized with a fusion protein of GST-C-terminal fragment of Prox-1protein. The antibody was affinity purified on the fusion protein andabsorbed with GST. The resulting antibody preparation (1.8 mg/ml) gave atiter of 1:10,000 against the fusion protein in ELISA.Immunofluorescence staining of tissue sections with the anti-Prox-1antibody gave a pattern of nuclear staining. Antibodies against thelymphatic markers anti-LYVE-1 (Laakkonen et al., 2002) andanti-podoplanin (kindly provided by T. Petrova and K. Alitalo), ratmonoclonal anti-mouse CD31 (BD Pharmingen), rat anti-mouse MECA-32(Pharmingen), rabbit polyclonal anti-T7 phage, and rat anti-mouse VEGFR3were used for immunohistochemical staining of frozen tissue sections asdescribed (Joyce et al., 2003; Laakkonen et al., 2002). Thecorresponding secondary antibodies were added and incubated for 1 hr atroom temperature: AlexaFluor-488 goat anti-rat or rabbit IgG (1:1000;Molecular Probes, Eugene, Oreg.), AlexaFluor-594 goat anti-rat or rabbitIgG (1:1000, Molecular Probes), AlexaFluor-594 donkey anti-mouse or goatIgG (1:1000, Molecular Probes), and AlexaFluor-488 donkey anti-mouse orgoat IgG (1:1000; Molecular Probes), respectively. The slides werewashed three times with PBS and mounted in Vectashield Mounting Mediumwith DAPI (Vector Laboratories, Burlingame, Calif.). Blood vessels werealso visualized by intravenously injecting Lycopersicon esculentum(tomato) lectin conjugated to fluorescein (100 μg of lectin in 200 μl ofPBS; Vector Laboratories). Tissue distribution of fluorescein-labeledpeptides (Laakkonen et al., 2004) was studied by intravenously injectingthe peptide (100-150 μgin 200 μl PBS) into the mice. The injectedpeptides were allowed to circulate 30 min to 2 hrs, and the mice wereperfused with 4% paraformaldehyde through the left ventricle of heart.Tissues were dissected and frozen in OCT embedding medium (Tissue-Tek,Elkhart, Ind.). The frozen sections were prepared for immunohistologicalanalysis.

Peptide Synthesis:

Peptides were synthesized using Fmoc chemistry in a solid-phasesynthesizer. The peptides were purified by HPLC and confirmed by massspectrometry. Fluorescein-conjugated peptides were synthesized asdescribed (Laakkonen et al., 2004). The LSD peptide and REA peptide weresynthesized as the chimera with the pro-apoptotic motif _(D)(KLAKLAK)₂(SEQ ID NO:19; Ellerby et al., 1999).

Transfection and Phage Binding Assay:

The 293T cells were transfected with plasmids encoding CXCR4 (Helbig etal., 2003) or VEGFR2 (Borges et al., 2000) using FuGene 6 transfectionreagent (Roche Diagnostics, Indianapolis, Ind.). Briefly, plasmid (10μg) was mixed with 900 μl of serumfree DMEM and 50 μl of FuGene andincubated for 15 min at room temperature before adding the mixture tothe cells. The cells were detached using EDTA after 48 hrs posttransfection and washed with PBS containing 1% BSA. Phage (5×10⁹ pfu)were incubated with the transfected cells and bound phage were rescuedand titrated. For competitive binding assay, the cognate peptide (150μg/ml) or antibody (50 μg/ml) was added during incubating of the phagewith cells. Targeted proapoptotic peptide treatment of tumor-bearingmice Prostate cancer model. Orthotopic xenografted prostate tumors wereestablished by injecting 1×10⁶ PPC1 human prostate cancer cells into themouse prostate. Fifteen days post inoculation, the mice wereintravenously injected with _(D)(KLAKLAK)₂-CREAGRKAC (SEQ ID NO:41), anequimolar mixture of _(D)(KLAKLAK)₂ (SEQ ID NO:19) and CREAGRKAC (SEQ IDNO:6), or PBS. Biweekly injections of 100 μg/dose/mouse were given forthree weeks. Melanoma model. Nude BALB/c mice were subcutaneouslyinjected with 1×10⁶ C8161 human melanoma cells. Treatment started whenmean tumor volumes reached about 100 mm3. Mice with size-matched tumorswere randomized into three groups. The therapeutic group received achimera of tumor homing peptide with the proapoptotic motif(_(D)(KLAKLAK)₂-CLSDCGKRKC; SEQ ID NO:42). The control groups receivedan equimolar mixture of CLSDGKRKC (SEQ ID NO:4) and _(D)(KLAKLAK)₂ (SEQID NO:19), or PBS alone. The tumor-bearing mice were intravenouslyinjected with 200 μg/dose/mouse once a week for three weeks. The micewere monitored for weight loss, and tumors were dissected and weighed atthe termination of the experiment. Histological analysis was performedto evaluate the density of tumor lymphatics and blood vessels. Theanimal experiments reported here were approved by The Burnham InstituteAnimal Research Committee.

Phage Overlay of Tissue Sections from Human Cancer:

The frozen sections of human prostate tumor specimens were obtained fromDr. Daniel Mercola (Sidney Kimmel Cancer Center, La Jolla, Calif.). Thesections (5 μm) were preincubated with blocking buffer (5% normal goatserum and 0.5% BSA in 1×PBS) for 1 hr at room temperature, washed threetimes with diluted blocking buffer (1:10), and phage (3×10⁹ pfu) wereincubated on the section for 4 hrs. After 3 washes, rabbit anti-phageantibody (10 μg/ml) was added and the phage incubated for 2 hrs. Theslides were washed and incubated with AlexaFluor-488 goat anti-rabbitIgG for 1 hr. After further washes, the slides were mounted withVectashield (VECTOR, Burlingame, Calif.).

Statistical Analysis:

Student's t test was used in statistical analysis of the results. Thebar diagrams show mean and standard deviation.

ii. Results

Phage Targeting of Lymphatics in C8161 Melanoma:

The C8161 human melanoma was chosen as the first target becausexenografts of tumors generated with this cell line in nude mice containlymphatic vessels that are not recognized by the homing peptide, LyP-1,which binds to lymphatic endothelial cells in breast carcinomas(Laakkonen et al., 2002). The experimental design was aimed to determinewhether lymphatic homing peptides having analogous specificity for themelanoma-associated lymphatics could be identified. protocols weremodified to increase the probability of obtaining peptides thatrecognize tumor lymphatics. A phage display library was incubated with acell suspension of whole C8161 tumor tissue, allowing phage to bind, andthen used immuno-magnetic beads to isolate lymphatic endothelial cellsthat carried along any phage bound to these cells. This enrichment stepyielded a phage pool that bound 250-fold more efficiently to theisolated cells than nonrecombinant phage (FIG. 9A). The enriched phagepool was used in subsequent in vivo rounds to select phage that homed toC8161 xenograft tumors. Two rounds of selection in vivo produced a40-fold enrichment of phage (FIG. 9B). There was no enrichment in theseveral control organs tested.

The 48 phage clones from the second in vivo round of phage poolselection included five clones that appeared most frequently, and thesewere analyzed further. Two clones displaying peptides with related aminoacid sequences CLSDGKRKC (SEQ ID NO:4) and CLDGGRPKC (SEQ ID NO:5) boundto cell suspensions prepared from C8161 tumors; the stronger binder,CLSDGKRKC (SEQ ID NO:4), bound 100 fold more than control phage.Intravenous injection of phage into nude mice bearing C8161 tumorsshowed that both phage homed selectively to the tumors; CLSDGKRKC (SEQID NO:4) was about twice as efficient as CLDGGRPKC (SEQ ID NO:5) (theresults for CLSDGKRKC (SEQ ID NO:4) are shown in FIG. 1A). The CLSDGKRKC(SEQ ID NO:4) peptide (referred to below as LSD) was chosen for furtherstudy. To establish that the homing ability of LSD phage is due to thedisplayed peptide sequence, the peptide was chemically synthesized as afluorescein-conjugate peptide and intravenously injected the conjugateinto C8161 tumor mice. After 2 hrs of circulation, the peptide wasdetected within the tumors, but not in control organs. Staining oftissue sections with the lymphatic vessel markers podoplanin, Prox-1,LYVE-1, and VEGFR3, showed co-localization of the LSD fluorescence withthem (FIG. 1C), whereas there was no co-localization with the bloodvessel markers MECA-32 and CD31. Quantification showed that 85% of thelymphatic vessels that were positive for the peptide were also positivefor podoplanin.

The homing of LSD phage to other types of cancer was further tested,including the MDA-MB-435 human breast cancer xenografts recognized bythe previously described lymphatic homing peptide, LyP-1 (Laakkonen etal., 2002). Intravenously injected LSD phage did not appreciably home toMDA-MB-435 tumors (see below). These data show that LSD-peptideselectively homes to the lymphatic vessels in C8161 melanoma.

FITC-LSD (150 mg) was intravenously injected into tumor mice and allowedto circulate for 2 hrs. The tumor and various tissues were collected andprocessed for histological analysis. A few spots of LSD fluorescencewere seen in the kidneys; all other tissues, with the exception of thetumors (FIG. 1B-D), were negative.

Phage Targeting of Lymphatics in Pre-Malignant Lesions and Tumors ofProstate:

Seeking to further generalize the proposition that tumor-associatedlymphatics might have organ specific signatures, lymphatic homingpeptides were selected in the TRAMP transgenic mouse model of de novoprostate carcinogenesis (Hsu et al., 1998). Immunohistochemical analysishad revealed abundant lymphatics associated both with pre-malignantlesions and tumors in this model. As it is possible to accesspre-malignant lesions in this system, the possibility of distinguishingthe lymphatics of such lesions from those of fully developed tumors wasalso explored.

To isolate peptides that selectively home to fully developed tumors inthe TRAMP model, the phage library were first pre-treated with cellsuspensions derived from normal prostate to decrease the abundance ofphage that bind to normal prostate. The normal prostate-subtractedlibrary was then enriched by two rounds of ex vivo selection onlymphatic endothelial cells immuno-purified from tumors of 25- to28-week-old TRAMP mice. Three subsequent in vivo selection roundsyielded a phage pool that showed nearly 50-fold enrichment for tumorhoming. Five peptide sequences were represented more than once in thispool. Three of these phage clones with amino acid sequences CREAGRKAC(SEQ ID NO:6), CSMSAKKKC (SEQ ID NO:7), and CKTRVSCGV (SEQ ID NO:8)showed robust binding to tumor derived cell suspensions and were furthertested in vivo. Intravenously injected CREAGRKAC (SEQ ID NO:6) phagebecame 50-fold enriched in TRAMP tumors relative to nonrecombinantphage, while the other two phage showed about 30-fold enrichment.CREAGRKAC (REA; SEQ ID NO:6) was chosen for further study.

To screen for peptides recognizing the pre-malignant lymphatics, thephage library was first treated with cell suspensions derived fromnormal prostate, and the subtracted library was then enriched onimmuno-purified lymphatic endothelial cell suspensions derived fromprostates containing pre-malignant lesions (14- to 16-week-old mice).The sequential ex vivo selections yielded a phage pool that was 60-foldenriched for binding to the target cells, and 30-fold enrichment forhoming to prostate with pre-malignant lesions was obtained in asubsequent in vivo selection. Five phage clones were chosen forevaluation of in vivo homing based on their frequent appearance among 64clones sequenced (32 clones each from the second ex vivo round and thethird in vivo round).

Of these, three clones with amino acid sequences CAGRRSAYC (SEQ IDNO:9), CASLSCR (SEQ ID NO:10), CSGGKVLDC (SEQ ID NO:11), bound to cellsuspension derived from pre-malignant prostate lesions. These candidateswere further tested in vivo individually. Phage displayed peptidesCAGRRSAYC (SEQ ID NO:9), CSGGKVLDC (SEQ ID NO:11), and CASLSCR (SEQ IDNO:10) showed 24-, 14-, and 12-fold enrichment to pre-malignant TRAMPlesions relative to nonrecombinant phage, respectively. CAGRRSAYC (AGR;SEQ ID NO:9) was chosen for further study.

To evaluate the specificity of the REA and AGR peptides, the phage wereintravenously injected into TRAMP mice with either pre-malignant lesionsor tumors, or into their tumor-free (transgene negative) malelittermates with normal prostates. The results showed that the REA phagehomes to tumors, but not to pre-malignant lesions or normal prostate(FIG. 2A), whereas the AGR phage homes only to pre-malignant lesions(FIG. 2B). Neither phage was found in other tissues, including lymphnodes, kidneys, lungs, skin, or gut, at levels higher than thenonrecombinant control phage.

In vivo distribution of fluorescein-conjugated REA and ARG peptidesafter intravenous injection confirmed the phage results. The REA peptideaccumulated in prostate tumors, showing 90% overlap withpodoplanin-positive lymphatic vessels, whereas premalignant lesions,normal prostate, or control organs were negative. The AGR peptideselectively homed to pre-malignant TRAMP lesions, but little or nopeptide was seen in prostate tumors, normal prostate tissue, or incontrol tissues.

Specifically, pre-malignant prostate tissue and tumor tissue wereobtained from TRAMP mice at the ages of 14-16 and 25-28 weeks,respectively. Lymphatics were visualized by staining frozen sectionswith rabbit anti-mouse LYVE-1 and blood vessels were stained with ratanti-mouse MECA-32. The fluorescein-labeled REA peptide (150 μg) wasintravenously injected into TRAMP tumor mice and the tumors and variouscontrol tissues were collected for histological analysis 2 hrs later. NoFITC-REA was detected in the skin, lungs, gut, or brain. The liver andkidneys contained fluorescence at levels far lower than the TRAMPtumors. The fluorescein-labeled AGR peptide (150 μg) was intravenouslyinjected into 14- to 16-week-old TRAMP mice and the tumors and variouscontrol tissues were collected for histological analysis 2 hrs later. NoFITC-AGR was detected in the skin, lungs, gut, brain, or heart. Thekidneys contained fluorescence at levels far lower than thepre-malignant lesions.

To study the association of REA and ARG peptides with the vasculature,the phage or the fluorescein-labeled peptides were intravenouslyinjected into TRAMP mice, and phage and peptide localization wascompared to lymphatic and blood vessel markers localized withantibodies. The phage and their cognate peptides each showed substantialco-localization with the lymphatic markers podoplanin, VEGFR3, LYVE-1,and Prox-1 in their respective lesions, whereas their localization wasentirely distinct from that of the blood vessel markers CD31 andMECA-32.

Homing Peptide for Lymphatic Vessels in Cervical Cancer:

A homing peptide for dysplastic skin lesions has been identified inK14-HPV16 transgenic mice, which develop skin cancers (Hoffman et al.,2003). This peptide, CNRRTKAGC (SEQ ID NO:17), is similar to LyP-1(CGNKRTRGC; SEQ ID NO:16), which selectively recognizes lymphaticvessels and tumor cells in breast cancers (Laakkonen et al., 2002).Because of this similarity, it was asked whether the CNRRTKAGC peptide(LyP-2; SEQ ID NO:17) also recognizes tumor lymphatics. The LyP-1 andLyP-2 peptides were tested in skin and cervical cancers of the K14-HPV16mice. In addition to their spontaneously developing skin cancers, femaleK14-HPV16 mice develop cervical cancers when treated with estrogen(Arbeit et al., 1996). These mice (K14-HPV16/E2 mice) develop tumors inboth organs through steps of neoplastic progression in a fashion thatmimics the human cancers (Arbeit et al., 1996; Coussens et al., 1996;Giraudo et al., 2004). The pre-malignant cervical lesions (also calledcervical intraepithelial neoplasia, CIN) and tumors of these micecontain abundant lymphatic vessels as detected by immunostaining forlymphatic markers.

LYVE-1 positive structures were seen in both the carcinoma and CIN-3lesion. Similar results were obtained with another lymphatic marker,Prox-1. Original magnification: 100×; inset, 400×. FIG. 11B showsfluorescein labeled-LyP-2 peptide homes to cervical carcinoma inK14-HPV16/E2 mice. FITC-LyP-2 peptide (100 μg) was injectedintravenously into tumor-bearing mice, and tissues were processed forhistological analysis 2 hrs later. Little or no fluorescence was seen innormal cervix, skin, liver, or brain.

Intravenously injected LyP-2 phage showed robust homing both to thepre-malignant and malignant lesions in the cervix, but not to normalcervix (FIG. 3). Fluoresceinlabeled LyP-2 peptide also accumulated inthe cervical lesions, co-localizing with LYVE-1 and podoplanin (82%overlap), but not with MECA-32. Additionally, occasional foci ofscattered cells in the stroma were labeled, with some apparentintracellular localization; the identity of these cells is currentlyunresolved. No peptide accumulation was observed in normal cervix or inother control tissues, either in lymphatics or in non-vascular cells(FIG. 11B). LyP-2 also homed to the lymphatics associated withdysplasias and squamous cell carcinomas in the skin in male and femalemice, but not to normal skin lymphatics.

Specificity of Lymphatic Homing-Peptides for Different Types of Tumors:

Having isolated phage-displayed peptides that homed to the lymphatics ofmelanoma, prostate, or cervix, it was asked whether they recognizedcommon determinants of the tumor-associated lymphatic vasculature ororgan/tumor selective signatures. The origin and specificity of thesepeptides is shown in Table 3. The lymphatic homing peptides derived fromthe different tumor models were tested for their ability to recognizethe lymphatics of other tumors. Intravenously injected LSD phage did nothome to xenotransplant tumors derived from the MDA-MB-435 breast tumorcell line (FIG. 4). This phage also did not appreciably home totransgenic mouse tumors of the breast or prostate, or to PPC1 humanprostate cancer xenografts; possible low-level homing was seen totransgenic skin cancers and KRIB human osteosarcoma xenografts. In vivoinjection of fluorescein-labeled LSD peptide followed by histologicalanalysis of peptide distribution agreed well with the phage results.Strong LSD peptide fluorescence was seen in the C8161 derived tumors,the model in which the peptide was selected. The C8161 tumors werepositive in nude mice representing two different genetic backgrounds(BALB/c and C57BL/6). In agreement with the phage data, KRIB tumors wereweakly positive with the fluorescent peptide, and the other tumors,including the skin cancers, were negative. These results show that theLSD peptide selectively recognizes the lymphatics in the C8161melanoma-derived tumors.

TABLE 3 Main characteristics of lymphatic homing peptides Fold overTumor used to isolate Tumors tested for phage homing in Specific controlPeptide homing peptide vivo* Homing^(†) phage LSD C8161 s.c. xenograftsC8161 xenografts Yes 39 KRIB xenografts Yes 7^(‡) K14-HPV16 skin cancerNo 5 MDA-MB-435 orthotopic No 3 xenografts MMTV-PyMT breast tumors No 3PPC1 orthotopic xenografts No 3 TRAMP prostate tumors No 1 REA TRAMPprostate TRAMP prostate tumors Yes 46 tumors PPC1 orthotopic xenograftsYes 25 M12 orthotopic xenografts Yes 24 LNCaP orthotopic xenografts Yes20 DU145 orthotopic xenografts Yes 14 MMTV-PyMT breast tumors Yes 8^(‡)K14-HPV16/E₂ cervical cancer Yes 7^(‡) KRIB xenografts Yes 7^(‡) PPC1s.c. xenografts No 6 C8161 s.c. xenografts No 5 K14-HPV16 skin cancer No4 MDA-MB-435 orthotopic No 4 xenografts AGR TRAMP PIN lesions TRAMP PINlesions Yes 18 TRAMP prostate tumors No 4 K14-HPV16/E₂ cervicaldysplasia No 5 K14-HPV16/E₂ cervical tumors No 4 MMTV-PyMT premalignantNo 2 lesions MMTV-PyMT breast tumors No 4 Lyp-2 K14-HPV16 skinK14-HPV16/E₂ cervical dysplasia Yes 17 cancer K14-HPV16/E₂ cervicaltumors Yes 22 MDA-MB-435 orthotopic No 3 xenografts *TRAMP, MMTV-PyMT,and K14-HPV16 are genetically engineered mouse models of organ-specificcarcinogenesis, each of which presents first with angiogenic dysplasiaand subsequently carcinoma. ^(†)The specific homing of phage isconsidered to be strong (>10-fold compared with control), weak (between5- and 10-fold), or non-specific (below 5-fold). ^(‡)Phage homingcorroborated by fluorescent peptide homing.

The REA phage, which was identified in the TRAMP model, also homed toxenografts obtained by orthotopically inoculating cells from the humanprostate cancer cell lines PPC1, M12, DU145, and LNCaP into nude mice(FIG. 5A). These xenografted tumors were also positive with thefluorescein-conjugated REA peptide. In contrast, the MDA-MB-435, C8161,and KRIB xenografts, as well as the de novo breast and skin cancersarising in MMTV-PyMT or K14-HPV16 mice, respectively, were negative forREA binding (FIG. 5A). The cervical tumors of K14-HPV16/E2 mice wereslightly positive for REA peptide binding, but markedly less so than theprostate tumors. Immunohistochemical analysis showed that FITC-REApeptide co-localized with lymphatic vessels in orthotopic prostate tumorxenografts arising from multiple human prostate tumor-derived celllines; this peptide homed to a lesser extent to K14-HPV16/E2 cervicaltumors. Interestingly, REA-phage homed less efficiently to subcutaneousxenografts of PPC1 than to orthotopic xenografts of the same tumor cellline (FIG. 10A). The REA-phage strongly bound to PPC1 tumor-derived cellsuspensions, but did not bind to cultured PPC1 cells (FIG. 9E). Thus,REA appears to primarily recognize prostate cancer lymphatics.

It was also evaluated whether the REA peptide recognizes human prostatecancers by using phage overlay of tissue sections. Immunohistochemicalstaining with antibodies against lymphatic markers Prox-1 and podoplaninrevealed abundant lymphatic vessels in human prostate tumors. Overlay oftissue sections from two primary human prostate cancers with REA phageindicated that this phage recognizes the lymphatics of human prostatetumors. The AGR phage did not bind to the human tumor sections.

To profile the homing peptide specificity of the AGR peptide indifferent types of premalignant lesions, three transgenic mouse modelswere used: TRAMP, K14-HPV16/E2, and MMTV-PyMT, which respectivelydevelop prostate, cervical, or breast neoplasias that subsequentlyprogress to overt cancer. Both AGR phage (FIGS. 2B and 5B) andfluorescent peptide showed marked preference for the TRAMP premalignantlesions; there was little homing of the phage and no detectable homingof the peptide to similar lesions or malignant tumors in the other twomodels (FIG. 5B).

LyP-1 and LyP-2 have Different Specificities:

Given the similar amino acid sequences of the LyP-1 and LyP-2 peptides,and the fact that they both bind to tumor lymphatics, theirspecificities were compared. Surprisingly, these peptides recognizedifferent tumors. While both peptides homed to the K14-HPV16 skin cancerlymphatics, LyP-1 phage homed to MDA-MB-435 tumors but not to thecervical tumors, whereas the opposite was true of LyP-2 (FIG. 6).Neither phage homed to the normal cervix or normal breast tissue. Toconfirm these differences in specificity, peptides were co-injected suchthat one peptide was a fluorescein conjugate and the other wasconjugated to rhodamine, and vice versa. Both LyP-2 conjugates homed tocervical tumors, whereas neither LyP-1 conjugate did so. The oppositeresult was obtained when the same conjugates were tested in MDA-MB-435tumor mice. These data indicate that different binding sites exist forthe two LyP peptides in different types of tumors.

Identification of a Candidate Homing Peptide Receptor:

Proteins with sequences homologous to the peptides can represent naturalligands for the receptor recognized by the peptide (Joyce et al., 2003).Database searches with the peptides described here revealed somehomologies (Table 4), one of which stood out: a homology of the LSDpeptide with the chemokine known as stromal cell-derived factor-1(SDF-1) or CXCL12 (FIG. 7A). CXCL12 is a ligand for the CXCR4 receptor.Transfecting 293T cells with CXCR4 cDNA rendered the cells capable ofbinding the LSD phage 16-fold more efficiently than mock-transfectedcells or cells transfected with VEGFR2 (FIG. 7B). The LyP-1 phage usedas a control did not bind to the CXCR4-transfected cells. The cognateLSD peptide inhibited the binding of the LSD phage to theCXCR4-transfected cells in vitro (FIG. 7C). These data indicate that theCXCL12/CXCR4 system is involved in the binding of the LSD peptide toC8161 lymphatics.

TABLE 4 Homing Peptide Homologies Mouse and human protein AccessionPeptide Peptide sequence Motif with the motif number LSD CLSDGKRKCC-SDGK mSDF-1 P40224 (SEQ ID NO: 4) (SEQ ID NO: 20) CLSDGK hSDF-1 P48061(SEQ ID NO: 2) LDG CLDGGRPKC CLDGG unknown (SEQ ID NO: 5)(SEQ ID NO: 21) REA CREAGRKAC GRKAC hCXCL1 P09341 (SEQ ID NO: 6)(SEQ ID NO: 22) CREA---AC hHGF-like protein precursor P26927(SEQ ID NO: 23) CREAG hIL-5R_ chain precursor, CD- Q01344,(SEQ ID NO: 24) MPR P20645 SMS CSMSAKKKC AKKKC mIL-17B Q9QXT6(SEQ ID NO: 7) (SEQ ID NO: 25) CS-S-KKK mSUT-1 Q9UKG4 (SEQ ID NO: 26)SMS-KK m IL-17Rh1 Q9JIP3 (SEQ ID NO: 27) KTR CKTRVSCGV KTRVS hEGF P01133(SEQ ID NO: 8) (SEQ ID NO: 28) AGR CAGRRSAYC CAGRR--S-YhNG3 (VE-statin 2), mNG3 Q99944, (SEQ ID NO: 9) (SEQ ID NO: 29) Q6GUQ1RRSAYC mType-1B angiotensin IIR P29755 (SEQ ID NO: 30) CAGR-SAmIL-22R_ chain Q80XF5 (SEQ ID NO: 31) RRSAY mCD1.2 P11610(SEQ ID NO: 32) RRS-YC mLeptin R P48356 (SEQ ID NO: 33) CAG-RS-Y hIL-27Q8TAD2 (SEQ ID NO: 34) ASL CASLSCR SLSCR mCD28 P31041 (SEQ ID NO: 10)(SEQ ID NO: 35) SGG CSGGKVLDC SG-KVLDC human integrin alpha-9 Q13797(SEQ ID NO: 11) (SEQ ID NO: 36) KVLDC Semaphorin receptor SEP O43157,(SEQ ID N0: 37) Q8CJH3 C-GG-VLD mouse uPAR P35456 (SEQ ID NO: 38) LyP-2CNRRTKAGC CNRR-K Arcadlin O95206 (SEQ ID NO: 17) (SEQ ID NO: 39) RR-K-GCKinesin-like protein KIF13A Q9EQW7 (SEQ ID NO: 40)

To identify mouse and human proteins with homologous sequences ofpeptides, peptides were analyzed by using a NCBI BLAST search againstthe SWISSPROT database with the option for short nearly exact matches.However, the LSD homology (CLSDGK; SEQ ID NO:2) spans the signal peptidecleavage site of pro-CXCL12 and thus is not represented in the maturechemokine (Nagasawa et al., 1994; Tashiro et al., 1993). Nevertheless,the induction of specific binding capability by transfection of CXCR4implicates this chemokine receptor, directly or indirectly, as a bindingtarget for the LSD lymphatic homing peptide.

Lymphatic Homing Peptide Conjugates Destroy Tumor Lymphatics:

Conjugates of vascular tumor-homing peptides with the apoptosis-inducingpeptide, _(D)(KLAKLAK)₂ (SEQ ID NO:19), are selectively cytotoxic toangiogenenic endothelial cells and have demonstrable anti-tumor activity(Ellerby et al., 1999). To determine whether peptides recognizing tumorlymphatics could be used to target those lymphatics, the REA and LSDpeptides were synthesized as conjugates with _(D)(KLAKLAK)₂ (SEQ IDNO:19) and systemically treated mice bearing PPC1 or C8161 xenografts.

Treatment with the REA conjugate reduced the number of tumor lymphaticsin the PPC1 tumors, whereas the uncoupled mixture had no effect comparedto the PBS control (FIG. 8A). The conjugate had no effect on tumor bloodvessel density (FIG. 8A) or tumor growth (FIG. 8B). Similar reduction oflymphatic vessel density was obtained in C8161 tumor mice treated withthe LSD conjugate.

H. References

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I. Sequences 1. SEQ ID NO: 1-hSDF-1 MNAKVVVVLVLVLTALCLSDGKPVS 2.SEQ ID NO: 2 CLSDGK 3. SEQ ID NO: 3 CLSDGKPVS 4. SEQ ID NO: 4-LSDCLSDGKRKC 5. SEQ ID NO: 5-LDG CLDGGRPKC 6. SEQ ID NO: 6-REA CREAGRKAC 7.SEQ ID NO: 7-SMS CSMSAKKKC 8. SEQ ID NO: 8-KTR CKTRVSCGV 9.SEQ ID NO: 9-AGR CAGRRSAYC 10. SEQ ID NO: 10-ASL CASLSCR 11.SEQ ID NO: 11-SGG CSGGKVLDC 12. SEQ ID NO: 12 KRTR 13. SEQ ID NO: 13RRTR 14. SEQ ID NO: 14 KRTK 15. SEQ ID NO: 15 RRTK 16.SEQ ID NO: 16-LyP-1 CGNKRTRGC 17. SEQ ID NO: 17-LyP-2 CNRRTKAGC 18.SEQ ID NO 18-LyP-3 CNKRTRGGC 19. SEQ ID NO: 19 _(D)KLAKLAKKLAKLAK 20.SEQ ID NO: 20 C-SDGK 21. SEQ ID NO: 21 CLDGG 22. SEQ ID NO: 22 GRKAC 23.SEQ ID NO: 23 CREA---AC 24. SEQ ID NO: 24 CREAG 25. SEQ ID NO: 25 AKKKC26. SEQ ID NO: 26 CS-S-KKK 27. SEQ ID NO: 27 SMS-KK 28. SEQ ID NO: 28KTRVS 29. SEQ ID NO: 29 CAGRR--S-Y 30. SEQ ID NO: 30 RRSAYC 31.SEQ ID NO: 31 CAGR-SA 32. SEQ ID NO: 32 RRSAY 33. SEQ ID NO: 33 RRS-YC34. SEQ ID NO: 34 CAG-RS-Y 35. SEQ ID NO: 35 SLSCR 36. SEQ ID NO: 36SG-KVLDC 37. SEQ ID NO: 37 KVLDC 38. SEQ ID NO: 38 C-GG-VLD 39.SEQ ID NO: 39 CNRR-K 40. SEQ ID NO: 40 RR-K-GC 41. SEQ ID NO: 41_(D)KLAKLAKKLAKLAK-CREAGRKAC 42. SEQ ID NO: 42_(D)KLAKLAKKLAKLAK-CLSDCGKRKC 43. SEQ ID NO: 43-mSDF-1MDAKVVAVLALVLAALCISDGKPVS 44. SEQ ID NO: 44-Antp RQPKIWFPNRRKPWKK 45.SEQ ID NO: 45-HIV-Tat GRKKRRQRPPQ 46. SEQ ID NO: 46-PenetratinRQIKIWFQNRRMKWKK 47. SEQ ID NO: 47-Antp-3A RQIAIWFQNRRMKWAA 48.SEQ ID NO: 48-Tat RKKRRQRRR 49. SEQ ID NO: 49-Buforin IITRSSRAGLQFPVGRVHRLLRK 50. SEQ ID NO: 50-TransportanGWTLNSAGYLLGKINKALAALAKKIL 51. SEQ ID NO: 51-model amphipathicpeptide (MAP) KLALKLALKALKAALKLA 52. SEQ ID NO: 52-K-FGFAAVALLPAVLLALLAP 53. SEQ ID NO: 53-Ku70 VPMLK-PMLKE 54.SEQ ID NO: 54-Prion MANLGYWLLALFVTMWTDVGLCKKRPKP 55. SEQ ID NO: 55-pVECLLIILRRRIRKQAHAHSK 56. SEQ ID NO: 56-Pep-1 KETWWETWWTEWSQPKKKRKV 57.SEQ ID NO: 57-SynB1 RGGRLSYSRRRFSTSTGR 58. SEQ ID NO: 58-Pep-7SDLWEMMMVSLACQY 59. SEQ ID NO: 59-HN-1 TSPLNIHNGQKL 60. SEQ ID NO: 60XRTX, where X is R or T

The invention claimed is:
 1. An isolated peptide having a length of upto 50 amino acids comprising CREAGRKAC (SEQ ID NO:6), wherein thepeptide selectively binds to tumor lymphatics.
 2. The peptide of claim1, wherein the peptide has a length of up to 25 amino acids.
 3. Thepeptide of claim 2, wherein the peptide has a length of from 9 to 25amino acids.
 4. A conjugate comprising the peptide of claim 1 conjugatedto a moiety, wherein the conjugate selectively homes to tumorlymphatics.
 5. A conjugate comprising the peptide of claim 1 and amoiety selected from the group consisting of a therapeutic moiety, adetectable moiety, a cytotoxic agent, an anti-lymphangiogenic agent, acancer chemotherapeutic agent, a pro-apoptotic polypeptide, a graftedpolypeptide, a virus, a cell, and a liposome, wherein the conjugateselectively homes to tumor lymphatics.
 6. The conjugate of claim 5,wherein the moiety is a cytotoxic agent, wherein the cytotoxic agent is_(D)(KLAKLAK)₂ (SEQ ID NO:19).
 7. The conjugate of claim 5, wherein themoiety is a detection moiety selected from a fluorophore, enzyme,biotin, metal, or epitope tag.
 8. A method of detecting cancer in asubject, comprising administering to the subject the conjugate of claim5 and detecting the presence of the conjugate in the lymphatics of thesubject, wherein detecting the presence of more of the conjugate in thelymphatics than a reference or control amount indicates the presence ofcancer.
 9. A method of detecting cancer, comprising contacting abiological sample with the conjugate of claim 5 and detecting thepresence of the conjugate in the lymphatics of the sample, whereindetecting the presence of more of the conjugate in the lymphatics than areference or control amount indicates the presence of cancer.
 10. Amethod of treating prostate cancer in a subject, comprisingadministering to the subject the conjugate of claim 5, wherein theconjugate inhibits lymphangiogenesis in a tumor in the subject.
 11. Themethod of claim 10, wherein the prostate cancer is malignant prostatecancer.
 12. A method of determining malignant prostate conditions in asubject, comprising contacting a biological sample from the subject witha conjugate according to claim
 4. 13. An isolated nucleic acid encodingthe polypeptide of claim
 1. 14. The isolated nucleic acid of claim 13further comprising a nucleic acid encoding a internalization sequence.15. The isolated nucleic acid of claim 14, wherein the cellularinternalization comprises an amino acid sequence of a protein selectedfrom a group consisting of Antennapedia, TAT, HIV-Tat, Penetratin,Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphipathicpeptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC(Bis-Guanidinium-Spermidine-Cholesterol and BGTC(Bis-Guanidinium-Tren-Cholesterol.
 16. An expression vector comprisingan isolated nucleic acid of claim 14, wherein the nucleic acid isoperably linked to an expression control sequence.